Hybrid phthalocyanine derivatives and their uses

ABSTRACT

Water soluble hybrid phthalocyanine derivatives useful in competitive and noncompetitive assays immunoassays, nucleic acid and assays are disclosed and claimed having (1) at least one donor subunit with a desired excitation peak, and (2) at least one acceptor subunit with a desired emission peak, wherein said derivative(s) is/are capable of intramolecular energy transfer from said donor subunit to said acceptor subunit. Such derivatives also may contain an electron transfer subunit. Axial ligands may be covalently bound to the metals contained in the water soluble hybrid phthalocyanine derivatives. Ligands, ligand analogues, polypeptides, proteins and nucleic acids can be linked to the axial ligands of the dyes to form dye conjugates useful in immunoassays and nucleic acid assays.

REFERENCE TO RELATED APPLICATIONS AND PATENTS

This application is a continuation of U.S. patent application Ser. No.09/776,599 filed Feb. 1, 2001, which in turn is a continuation of U.S.patent application Ser. No. 09/066,255 filed Apr. 24, 1998, which issuedas U.S. Pat. No. 6,964,844 on Nov. 15, 2005, which in turn is acontinuation of U.S. patent application Ser. No. 08/620,597, filed Mar.22, 1996, which issued as U.S. Pat. No. 5,824,799 on Oct. 20, 1998,which in turn is a continuation-in-part of U.S. patent application Ser.No. 08/409,825, filed Mar. 23, 1995, now abandoned, and of U.S. patentapplication Ser. No. 08/311,098, filed Sep. 23, 1994, which issued asU.S. Pat. No. 5,763,189 on Jun. 9, 1998, and of U.S. patent applicationSer. No. 08/274,534, filed Jul. 12, 1994, which issued as U.S. Pat. No.6,238,931 on May 29, 2001, and of U.S. patent application Ser. No.08/138,708, filed Oct. 18, 1993, now abandoned, and of U.S. patentapplication Ser. No. 08/126,367, filed Sep. 24, 1993, now abandoned,from each of which priority is claimed, and each of which are herebyincorporated by reference herein and for all purposes.

TECHNICAL FIELD

This invention relates generally to the synthesis of novel dyes andlabels and methods for the detection or visualization of analytes andmore specifically to fluorescent latex particles which incorporate thenovel fluorescent dyes and utilize, in certain aspects, fluorescenceenergy transfer and intramolecular energy transfer, for the detection ofanalytes in immunoassays or in nucleic acid assays.

BACKGROUND ART

Various methodologies are available for the visualization of cells ormolecules in cells and for the measurement of analyte concentrations influids. Fluorescence microscopy utilizes fluorescent dyes, generallyconnected to specific probes, such as antibodies, for the localizationof proteins and complexes in cells. For the measurement of analyteconcentrations, immunoassays have become popular over the last 40 yearsbecause of the specificity of antibodies toward the analyte or targetligand. Radioimmunoassays were developed because the high specificactivity of the radionucleotide allowed measurement of very lowconcentrations of analyte. However, because of the concerns for theenvironment and human health, the use of radionucleotides inimmunoassays is becoming less popular. The use of enzymes inimmunoassays to amplify a signal has been a very important advance inthe field of immunoassays because their use does not involveenvironmental or human health hazards or risks. Enzyme-linkedimmunoassays, however, can be problematic because the activity of theenzyme is temperature dependent and the instability of the enzyme or thesubstrates can result in inaccurate quantitation of the target ligand.Still other immunoassays monitor fluorescence as the signal, with orwithout enzymes, for the measurement of analyte concentrations.

The characteristics of the fluorescent dyes are very important whenquantifying analyte concentrations in biological fluids. For example,when the biological fluid is blood, serum or plasma, the intrinsicfluorescence of the fluid precludes the use of many dyes. Thesebiological fluids generally have fluorescence emissions up to 600 nmwhen exciting at various wavelengths above 200 nm. The fluorescence isgenerated by excitation of the dye at the appropriate wavelength. Thefluorescent signal is measured by a fluorometer which is tuned to excitethe fluorescent molecule at a specific wavelength and to measure theemission of fluorescence at another wavelength. The difference in theexcitation and emission wavelengths is referred to as the Stokes shift.To achieve the most sensitive measurement, the emission wavelength ofthe sample should not interfere with the emission of the dye. Also, theStokes shift should be as large as possible so that the excitation lightis not seen by the detector as a background signal. When the Stokesshift is not large, filters or monochromators can be utilized in thefluorometer to exclude light near the emission wavelength; however, theuse of filters decreases the yield of light reaching the detector andgenerally one circumvents this problem of light loss by the use of highintensity lamps. Thus, to avoid problems associated with small Stokesshifts and dyes which emit near the intrinsic emission of the biologicalfluid, a sophisticated instrument is generally built. With the advent ofnear-patient diagnostics in hospitals, there is a need for portable,simple fluorometers which can assess fluorescence in an immunoassay forthe detection of analytes in biological samples.

Another problem associated with the assay of analytes in fluids or thevisualization of cellular components with an intrinsic fluorescence isthat of selection of the dye which is utilized as the label. The dye isgenerally chosen for its brightness (the product of fluorescence quantumyield and extinction coefficient) since a certain sensitivity in theassay or the visualization technique is required. However, the selectionof the dye used as the label is limited when the sample has an intrinsicfluorescence because the instrument may not be capable of distinguishingsample fluorescence from dye fluorescence.

The current invention provides a methodology for the development ofamplified fluorescent label systems which can be tuned to specificexcitation and emission wavelengths. The methodology teaches improvedmethods for incorporation of dyes into particles to minimizefluorescence quenching and to maximize fluorescence intensities of thedye molecules in the particles. In addition, the design and synthesis ofnovel hybrid phthalocyanine derivatives are described which areincorporated into particles or are synthesized as water-solublemolecules for use as labels and are directly coupled to proteins,polypeptides, other labels, nucleic acids and the like. The novel dyesystems can be utilized for the quantitation of analytes in fluids, andin particular, in biological fluids. The novel dye systems can be tunedto specific exciting and emitting wavelengths so that low currentsources, such as light emitting diodes and laser diodes, and detectors,such as photo diodes, and the like, can be used in the manufacture offluorometers which can be battery powered and portable, for use, forexample, in immunoassays dedicated to near-patient diagnostics.

DISCLOSURE OF THE INVENTION

This invention relates to novel fluorescent particles and novel watersoluble fluorescent dyes. These novel particles and dyes can be tuned tospecific excitation and emission wavelengths to accommodate a widevariety of assay or visualization systems. In yet another aspect of theinvention, the methodology teaches improved methods for incorporation ofdyes into particles to minimize fluorescence quenching and to maximizefluorescence intensities of the dye molecules in the particles throughthe use of different dye molecules which possess the same or verysimilar excitation and emission wavelengths.

Many novel phthalocyanine derivatives and hybrid phthalocyaninederivatives are disclosed and claimed. In one embodiment microparticlesare disclosed having at least one hybrid phthalocyanine derivative, saidderivative(s) having (1) at least one donor subunit with a desiredexcitation peak; and (2) at least one acceptor subunit with a desiredemission peak, wherein said derivative(s) is/are capable ofintramolecular energy transfer from said donor subunit to said acceptorsubunit.

In another embodiment, water soluble hybrid phthalocyanine derivativesare disclosed having (1) at least one donor subunit with a desiredexcitation peak; and (2) at least one acceptor subunit with a desiredemission peak, wherein said derivative(s) is/are capable ofintramolecular energy transfer from said donor subunit to said acceptorsubunit. Such derivatives also may contain an electron transfer subunit.Axial ligands may be covalently bound to the metals contained in thehybrid phthalocyanine derivatives. The axial ligands of the dyes can befurther elaborated with drug analogues and compounds, proteins,polypeptides and nucleic acids. Numerous compounds capable ofintramolecular energy transfer as well as compounds for fluorescenceenergy transfer are claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structures of phthalocyanine, naphthalocyanine andanthranylocyanine.

FIG. 2 depicts the structures of silicon phthalocyanine, siliconnaphthalocyanine and silicon anthranylocyanine.

FIG. 3 depicts the spectra of silicon phthalocyanine dihydroxide and thespectra of silicon 2,3-naphthalocyanine dihydroxide.

FIG. 4 depicts the general structure of ethenyl-substituteddipyrrometheneboron difluoro dyes.

FIG. 5 depicts the attenuation of the background signal as a function ofincreasing wavelength. The data was measured using a device as describedin Applicant's allowed Ser. No. 07/887,526 filed May 21, 1992 entitled“Diagnostic Devices and Apparatus for the Controlled Movements ofReagents Without Membranes,” which is hereby fully incorporated herein.

FIG. 6 depicts naphthalocyanine derivatives which emit in the nearinfrared.

FIG. 7 depicts general structures of fluorescent energy transfernaphthalocyanine compounds.

FIG. 8 depicts the absorbance spectrum of human serum between 200 nm and1000 nm.

FIG. 9 depicts the structure of a novel hybrid phthalocyaninederivative, Silicon [di(1,6-diphenylnaphthalocyanine)]diphthalocyaninebis(dimethythexylvinylsilyloxide).

FIG. 10 depicts the spectrum ofSilicon[di(1,6-diphenylnaphthalocyanine)]diphthalocyaninebis(dimethythexylvinylsilyloxide).

MODES FOR CARRYING OUT THE INVENTION

This invention describes novel fluorescent particles and novelfluorescent molecules and diagnostic methods for their use. Developing amethod for the visualization of a cellular component or a cell or for anassay which utilizes a fluorescent dye and which quantifies an analytein a sample requires the use of a fluorometer. The fluorescent label,the sample and the instrument must be compatible with each other toachieve an accurate measurement. Several criteria for a fluorescentlabel as they relate to the sample and instrument are described below.First, the absorption or excitation and emission wavelengths of the dyeshould not correspond so closely to the absorption or fluorescence ofthe specimen or sample such that the sample affects the fluorescencemeasurement of the dye. Second, the Stokes shift of the dye should be aslarge as possible to minimize the measurement of background from theexcitation wavelength. Third, the dye must be compatible with the phaseof the visualization or the fluid phase of the assay; that is, the dyemust be water soluble or water insoluble depending on the visualizationor assay format. Fourth, the dye should be as bright as is necessary toachieve the desired sensitivity. Brightness is the product of theextinction coefficient and the quantum yield of the dye. Fifth, theinstrument used to detect the fluorescent signal is generally designedaround the specifications of the dye and the specimen or sample beingvisualized or assayed.

These points will be discussed in more detail and illustrate some of theintricacies in developing a fluorescent visualization technique or anassay using fluorescent dyes. One is limited either to dyes which havebeen synthesized or ones which must be synthesized in order to meet theabove criteria. Using prior art methods, a very limited range ofexcitation and emission wavelengths can be planned for a specificmolecule. The teachings of this invention allow one to preparefluorescent dyes and labels which can be tuned to many excitation andemission wavelengths allowing for large Stokes shifts. Thus, designing adye system with the specifications of the sample or specimen and theinstrument is possible from the teachings of this invention, as opposedto the prior art methods which involve designing the instrument aroundthe specifications of the dye. Tuning the dye system to accommodate thecharacteristics of the sample and the instrument results in an improvedvisualization process for the assay.

The excitation and emission wavelengths of the dye should not correspondto those of the sample being assayed or visualized, otherwise the samplecan interfere with the measurement of the fluorescent signal. Whenabsorption or emission wavelengths of the sample do correspond to thoseof the dye, in practice, one dilutes, for example, a serum or bloodsample so that the interference by the sample is reduced or theinterfering sample is washed away from the detection area. Indeed,currently, there is no fluorescent assay system on the market for themeasurement of analytes in neat biological fluids, particularly blood,plasma or serum. One reason for the lack of fluorescent assay systemswhich detect analytes in neat samples is that no good fluorescent dyeexists which meets all the criteria listed above, particularly formeasuring fluorescence in biological samples. When the sample absorbssignificantly at the excitation wavelength the amount of light whichexcites the sample is thus affected by the variation in the samplecharacteristics. For example, serum, plasma, or blood from differentindividuals will be different in their relative absorptivities, whichdifferences translate into different intensities of excitation lightused to excite the fluorescent label. The fluorescence emission of thedye is directly proportional to the intensity of the incident light,such that when the sample absorbs a portion of the incident light, theintensity of the fluorescent signal will vary accordingly. This resultsin measuring an incorrect or effected fluorescence emission. Inaddition, the emission wavelength of the dye should not correlate withthe emission or absorbance of the sample because the sample willincrease the measured fluorescence of the dye or the sample will absorball or a portion of the dye fluorescence and also result in an incorrector effected fluorescence emission. These problems are avoided when thesample is invisible to the excitation and emission wavelengths.

FIG. 8 shows the spectrum between 200 nm and 1000 nm of human serum.Wavelengths above 600 nm absorb considerably less than those between 200nm and 600 nm. Thus, both the absorption of the incident light and theeffect on the fluorescence of a dye are minimal when exciting above 600nm. Preferred excitation wavelengths for biological fluids, includingurine, blood, serum or plasma is 600 nm or greater. Particularlypreferred excitation wavelengths above 600 nm are those which correspondto the maximum light output of laser diodes and light emitting diodes.Preferred emission wavelengths are those above 600 am. The intrinsicsample fluorescence can cause a high background signal if the emissionwavelength of the dye and the sample are overlapping. In addition, thescattered light of the excitation source can also contribute to thebackground signal. The contribution of scattered light to the backgroundcan be seen, for example, in FIG. 5. In general, the magnitude of thescatter is inversely proportional to the fourth power of the measuredwavelength. This teaches that desired emission wavelengths are in thenear-infrared or in the infrared region of the spectrum. The inventiveteachings described herein provide for dyes and dye systems which exciteabove 600 nm and which emit above 650 nm and more preferred, above 730nm.

The Stokes shift of the dye should be as large as possible to minimizethe measurement of background from the excitation source so that thesignal-to-background ratio at the limit of sensitivity is maximized. Alarge Stokes shift, however, will only maximize the efficiency of thefluorescence measurement and may not always result in an accuratefluorescence measurement. For example, table 3 shows data from severaldye systems which were excited between 420 nm and 670 nm in eitherbuffer, undiluted human serum and blood. The fluorescence intensity ofthe first dye system (line 1, table 1), when excited at 475 nm in serumand blood, is only 7.6% and 13%, respectively, of the intensity inbuffer even though the Stokes shift is 205 nm. The second dye system(line 4, table 1), excited at 420 nm, is 28% and 4% in serum and bloodof the intensity in buffer, respectively, with a 260 mm Stokes shift.The third and fourth dye systems (line 60 and line 59, table 1), excitedat 670 nm and 650 nm and with 110 nm and 130 nm Stokes shifts,respectively, have fluorescence intensities which are comparable inbuffer and in serum. The fifth dye system (line 107, table 1), excitedat 670 nm with a 90 nm Stokes shift, has fluorescence intensities whichare also comparable in buffer, serum and blood. The sixth dye system,which is a hybrid phthalocyanine derivative (Line 1, table 2), hascomparable fluorescence intensities in buffer, serum and blood whenexcited at 646 nm with a Stokes shift of 114 nm. The data show that thefluorescence intensity is greatly affected when the excitationwavelength is within the range of the absorbance of the sample in whichthe measurement is made. The data also show that the magnitude of theStokes shift does not have an influence on the accuracy of themeasurement. These data are representative of other dyes and dye systemswhich are excited at a wavelength where the sample absorbs. The effectof the decreased fluorescence emission is not a result of the emissionwavelength (that is, 680 nm or 780 nm) because the samples absorbminimally at 680 nm and 780 nm. One skilled in the art can appreciate,that with the inventive teachings described herein, the wavelengths forexcitation and emission of a dye system should be a function more of theabsorption and emission characteristics of the sample rather thanselecting only a dye system with a large Stokes shift.

The availability of dyes with Stokes shifts greater than 100 nm isgreatly limited, particularly when the excitation wavelength is greaterthan 600 nm. To further limit the usefulness of available dyes, thesolubility of the dyes in aqueous samples can be a problem because mostdyes with large Stokes shifts are water insoluble.

The problem of a dye possessing a small Stokes shift is usually overcomein the engineering of the fluorometer by the use of monochromators orexpensive optics which filter out the light from the excitation source.However, to overcome the loss in light intensity due to the filters, forexample, one requires the use of high powered light sources. These lightsources produce heat which must be dissipated in an instrument by usingheat sinks or fans. The complexity of the fluorescence measuring device,both from an optical and a mechanical perspective, is thus greatlyaffected by the inadequacies of the dye system. With the advent ofnear-patient testing in hospitals and emergency departments, instrumentswhich measure fluorescence in immunoassays will be required to beportable and uncomplicated to the technician. Thus, the future state ofthe art for the manufacture of, for example, fluorometers which areemployed for immunoassays will be required to change to simple andportable instruments. The high powered light sources and expensiveoptics currently incorporated into fluorometers will not meet therequirements for small, portable instruments.

The instant invention teaches that fluorescent labels can be preparedwith large Stokes shifts and be tuned to wavelengths both of which arecompatible with excitation sources and emission detectors and which arecompatible with the absorption and emission of the sample, for example,blood, serum, plasma, urine, ground water, and the like. The excitationand emission wavelengths of the novel fluorescent dyes and particles cangenerally be varied independently of each other.

The dye must be compatible with the fluid phase of the assay, or inother words, the dye must be water soluble or water insoluble dependingon the visualization or assay format. Many fluorescent dyes are waterinsoluble or poorly water soluble and these dyes are not easily used forlabeling molecules, proteins, nucleic acids or cells. One skilled in theart will recognize that water insoluble dyes can be incorporated intolatex particles as described in U.S. Pat. Nos. 4,326,008, 4,609,689 and5,154,887, which are hereby incorporated by reference. Thus, waterinsoluble dyes can be made useful by incorporation into latex particlesfor visualization in a variety of assay formats.

The dye should be as bright as is necessary to achieve the desiredsensitivity. If one knows the extinction coefficient and the quantumyield of the dye and the concentration of the target to be measured, itcan be estimated whether the dye is bright enough to achieve the desiredsensitivity. Incorporation of dyes into latex particles or theutilization of an enzyme which catalyzes the production of a fluorescentsubstrate are examples of techniques which one skilled in the art usesas amplification systems.

The instrument used to detect the fluorescent signal is generallydesigned around the specifications of the dye and the specimen or samplebeing visualized or assayed because of the limited numbers of dyes whichcan be successfully used. As discussed above, the components of theinstrument are selected for a particular dye system since a usefulinstrument must be highly tuned to eliminate the light from theexcitation source.

Each of the conditions described above impose limitations on dye systemswhich can be employed for measuring sub-picomolar concentrations ofanalytes, particularly in biological fluids. The limitations also imposerestrictions on the design of an instrument to measure the fluorescence.The novel teachings of the instant invention allow the design, synthesisand tuning of dye systems to match, generally, nearly any instrumentdesign.

Several inventive teachings are described for tuning excitation andemission wavelengths of dyes so that the excitation and emission arecompatible with the sample matrix in which the fluorescence is measuredand the instrument for quantifying the fluorescence. One teaching is toeither incorporate or adsorb at least two dyes into or onto particles,which, as a pair, exhibit fluorescence energy transfer. The particleswhich can be used are those which adsorb dyes on the surface or absorbor imbibe dyes inside the particle. Another teaching is to incorporatedyes which are covalently attached to each other and which also exhibitfluorescence energy transfer both in solution and in particles.

Another teaching is to incorporate hybrids of phthalocyanines,naphthalocyanines, anthranylocyanines (collectively termed hybridphthalocyanine derivatives) and various derivatives of these classes ofcompounds which have different subunits depending on the desiredexcitation or emission wavelengths. The hybrid phthalocyaninederivatives may also be synthesized as water soluble compounds to beused for direct attachment to proteins, polypeptides other labels ornucleic acids. One advantage of hybrid phthalocyanine derivatives isthat they allow one to create dyes and dye systems which have greaterStokes shifts with higher extinction coefficients at the excitationwavelength. This is accomplished by properly selecting the subunitswhich are to be tetramerized to form the hybrid phthalocyaninederivative structure and which will absorb the light at the excitationwavelength.

The selection of dye pairs for incorporation into particles is based ontheir ability to exhibit energy transfer (singlet-singlet energytransfer) at the appropriate excitation wavelength of the donor dye andthe emission of the acceptor. Fluorescence energy transfer of twomolecules is well known to those skilled in the art and the rate ofenergy transfer is described by Forster in Ann. Physik. (1948) 2, 55-75.Fluorescence energy transfer has been used as a spectroscopic ruler topredict proximity relationships in proteins, RNA and peptides (AnnualReview of Biochemistry (1978), 47, 819-846) and also to probegeometrical details in particles (Physical Review Letters (1988) 61,641-644). U.S. Pat. No. 5,326,692 describes fluorescent particles withcontrollable enhanced Stokes shifts. U.S. Pat. Nos. 4,542,104 and4,666,862 describe fluorescence energy transfer in phycobiliproteins.These dye complexes are described for use as labels in immunoassays. Thelimited use, however, of phycobiliproteins and the expense of thesenatural protein complexes make them undesirable for use on a commercialscale. Some unsymmetrical or hybrid phthalocyanines have been described,for example, in J. Am. Chem. Soc. 1990, 112, 9640-9641, ChemistryLetters 1992, 2031-2034 and Inorg. Chem. 1994, 33, 1735-1740, but thisinvention greatly expands the compounds which can be synthesized for usein immunodiagnostics to achieve adequate fluorescence intensities anddesired excitation and emission characteristics. The ratio of thevarious diiminoisoindiline or dicarbonitrile precursors and theirsubstitution by electron donating or electron withdrawing groups in thesynthesis of the hybrid phthalocyanines, naphthalocyanines andanthranylocyanines will affect the absorption spectrum and theexcitation and emission wavelengths of the compounds. This is taught andapplied to the novel dyes herein.

In one aspect, the novel fluorescent particles of this invention arecomposed of at least two dyes which are positioned in the interior or onthe exterior of particles at an energy exchanging distance. One skilledin the art will recognize that various particles can be utilized, suchas latex, silica, alumina, liposomes, various colloids and the like.Particularly preferred particles are latex particles. The selection ofthe dye molecules for incorporation into the particles should be relatedto the specific use of the particles, the sample to be analyzed and theinstrument for measuring the fluorescence. For example, when developingan assay for an analyte in a biological medium, such as blood, serum ora cell extract, the intrinsic absorbance and fluorescence of the samplemust be considered. Serum and cellular components absorb in theultraviolet spectrum as well as in the visible spectrum up to around 600nm and the intrinsic fluorescence can broadly approach 600 nm. Inaddition, samples which contain small particles, such as dirt particlesin ground water, lipoproteins in serum or blood, cells and cellularparticles and components will scatter the excitation light which resultsin a higher background signal. The ideal dye couple would include thedonor dye which would be excited or absorb at above 600 nm and emit at awavelength which the acceptor dye absorbs, and the acceptor dye shouldemit at a wavelength above 600 nm. In the case of a single dye system,for example, with the use of hybrid phthalocyanine derivatives, theexcitation and emission wavelengths should also be above 600 nm. Thesample, for example, serum, then does not affect fluorescence of theacceptor dye because the sample poorly absorbs at the absorption of thedonor dye and the acceptor dye emits at a wavelength where the sampledoes not absorb or fluoresce.

Fluorescent dye molecules incorporated into or onto particles willexhibit fluorescence quenching because of the close proximity of thedyes to each other and to the matrix of the particle. When loading dyesinto or onto particles, one must optimize the concentration of dye as itrelates to quenching. The dyes can be loaded successively or together.The degree of quenching can be quantified by measuring the fluorescenceemission of a dilute suspension of particles (about 0.001% to 0.1%solids) in a buffer solution, in a buffered protein solution or in waterand then also measuring the fluorescence of the same concentration ofparticles in solvent which liberates the dyes from the particles. Theratio of the fluorescence intensities (1-[fluorescence intensity ofincorporated dyes divided by the intensity of liberated dyes] is thedegree of quenching of the dyes in the particle. In practice, oneincorporates dyes at various concentrations and measures thefluorescence intensities of the incorporated and liberated dyes tooptimize the intensity of fluorescence of the particle while minimizingthe quenching of fluorescence in the particle. In a situation where morethan one acceptor dye is used to minimize fluorescence quenching and tomaximize fluorescence intensity, one may use different acceptor dyeswhich have emission peaks which are within about 25 nanometers of oneanother. The emission of both acceptor dyes may be useful if thefluorometer is set-up to measure a wide band pass of fluorescence, forexample, about a 20 to 60 nm bandpass.

Another important consideration is the efficiency of the fluorescenceenergy transfer. In practice, if the energy transfer efficiency is notclose to 100%, then one can observe the fluorescence of the donor dye.The resulting fluorescence of the donor dye can make the particlesundesirable or even useless because the “effective Stokes shift” (thatis, the shortest wavelength distance to a light source from the definedacceptor molecule emission wavelength in the fluorescence system) of theparticles is now not the difference between the excitation and emissionwavelengths of the donor and acceptor dyes, respectively, but rather thedifference between the donor emission and the acceptor emissionwavelengths. The emissions of the donor and acceptor wavelengths canoverlap partially with each other when efficient energy transfer is notobtained and complicate the selection of filters for use in afluorometer. The decrease in the energy transfer efficiency can also bedirectly related to a decrease in the emission of the acceptor dye,resulting in a particle which may not be as bright as a particle withefficient energy transfer. In addition, under conditions of inefficientenergy transfer, slight changes in the sample or in solution conditions,for example, pH, ionic strength and the like, may affect the magnitudeof energy transfer efficiency and thereby may affect the intensity ofthe fluorescent signal.

In selecting dye pairs for fluorescence energy transfer one begins bystudying the overlap of the donor emission and acceptor excitationwavelengths. The dyes are positioned in the particle at an energyexchanging distance from one another which allows singlet-singlet energytransfer. Although a particular pair of dyes has acceptable overlappingexcitation and emission wavelengths (for example, see Proc. Natl. Acad.Sci. USA 1969, 63, 23-30), they may not exhibit fluorescence energytransfer in particles or they may have suboptimal (less than 80%)efficiency of energy transfer. The process to determine whether 2 ormore dyes will exhibit efficient energy transfer is throughexperimentation after the appropriate spectral overlap criteria are met.The efficiency of fluorescence energy transfer is determined bymeasuring the fluorescence intensity of the donor dye alone in particlesand also measuring the fluorescence emission of the particles which haveincorporated 2 or more dyes (that is, the fluorescent energy transferparticle) at the emission wavelength of the donor dye, both sets ofparticles having the same concentrations of donor dye and particles. Themeasured fluorescence at the donor dye emission wavelength of thefluorescent energy transfer particles divided by the fluorescence of thedonor dye particles is the efficiency of fluorescence energy transfer.Ideally, in practice, the emission of the donor dye should beundetectable or only slightly detectable so that the effective Stokesshift is not reduced because of the donor dye emission. Preferredfluorescence energy transfer efficiencies are 80% or greater andparticularly preferred fluorescence energy transfer efficiencies are 90%or greater.

Another important criteria for preparing particles exhibitingfluorescence energy transfer is the selection of the solvent used toswell and/or imbibe the dyes. The solvent system should penetrate theinterior of the particle, for example, when using latex particles, andthe dyes should also be soluble in the solvent system so that the dyesin the solvent can enter the interior of the particle. Optimization byexperimentation is recommended, however, to produce particles withenergy transfer or with optimum energy transfer. For example, table 6 ofExample 67 shows the results of fluorescence energy transfer in latexparticles prepared with dimethylformamide and tetrahydrofuran, both ofwhich swell latex particles and dissolve the dyes.

When using particles which are not porous, for example, silica oralumina, for fluorescence energy transfer, the solvent system shoulddissolve the dyes but allow the dyes to adsorb to the particles. In someinstances, it may be necessary to exchange solvent systems to adsorb thedyes; that is, the first solvent system dissolves the dyes in theparticle slurry and a second solvent is introduced which promotes theadsorption of the dyes to the particles. When preparing liposomes whichcontain energy transfer dyes, ultrasonic techniques, for example, can beutilized to trap the dyes in the liposome interior as the liposome isformed, Techniques for forming liposomes can be found in, for example,Liposome Technology, Volumes I-III (1984), ed., G. Gregoriadis, CRCPress Inc.

The novel particles described herein exhibit reduced quenching andimproved fluorescence intensities. A large majority of fluorescentmolecules have aromatic character, that is, they possess 4n+2πelectrons. The resultant aromatic character promotes stacking of themolecules, especially of water insoluble molecules in aqueous solutionsor in particles in aqueous solution, which in turn promotes fluorescencequenching. The novel particles described herein are incorporated withdyes which, through steric interference of the dye molecules, have aminimized propensity to stack in the particles.

In another aspect of this invention, fluorescence quenching of dyemolecules in particles is minimized by employing different dyes withapproximately the same excitation and emission wavelengths. That is, thewavelength maximum for excitation and/or emission of the different dyesis within about 25 nm of each other so that there is substantial overlapof the peaks. Different dyes will not stack in an organized orientationwith each other to the same degree as dyes which are the same.Incorporating different dyes into or onto particles using organicsolvents and then removing the solvent causes the dye to precipitate orcrystallize in the particle. The disruption of the crystalline latticeof dye molecules in particles alters the stacking of the molecules andthereby reduce quenching. Thus, incorporation of dissimilar dyemolecules with similar excitation and emission spectra improvesfluorescence intensities of the particles by decreasing the quenchinginteractions of the molecules.

In another aspect of this invention, incorporation into particles ofdissimilar dyes which exhibit fluorescence energy transfer in theparticles may also disrupt the other's crystalline lattice formation.Thus, the fluorescence intensities of particles exhibiting fluorescenceenergy transfer will be improved as a result of decreasing quenching inthe particle because the stacking of similar dyes in the particles isdisrupted by the dissimilar dye.

In yet another aspect of this invention, the synthesis of phthalocyaninederivatives and hybrid phthalocyanine derivatives with axial ligandsreduces the stacking of the aromatic ring system, thus minimizing theinteractions between molecules and maximizing fluorescence intensities.

One skilled in the art can appreciate that more than one dye pair whichexhibits fluorescence energy transfer can be incorporated into or ontoparticles resulting in a class of particles which fluoresce at differentwavelengths. In addition, with the inventive teachings described herein,incorporation into or onto particles of 3 or more dyes, which togetherprovide a cascade of energy transfer from the absorber(s) to theintermediate donor(s) to the acceptor(s) (which fluoresces), can resultin the production of particles with very long Stokes shifts and allowsone to produce particles with nearly an unlimited variety of excitationand emission characteristics.

FIG. 1 shows preferred acceptor dyes which are phthalocyanines,naphthalocyanines and anthranylocyanines. FIG. 2 shows particularlypreferred acceptor dyes which are derivatives of siliconphthalocyanines, naphthalocyanines and anthranylocyanines, where R ishydrogen or an alkylcarbon chain from 1-20 carbons, either saturated orunsaturated, having 0-10 heteroatoms (N, O, S), and having 0 or 1siloxide groups. The best mode compounds are those in which R=

Si(CH₃)₂C₆F₅

Si(C₆H₁₃)₃

Si(CH₃)₂(CH₂)₃CN

Si(CH₃)₂(CH₂)₁₀COOCH₃

Si(CH₃)₂CH═CH₂

Si(CH₃)₂(CH₂)₁₀COOH

Si(CH₃)₂(CH₂)₄Cl; and

Si(CH₃)₂(CH₂)₆CH═CH₂.

The parent compounds of the phthalocyanines and naphthalocyanines arepreferred because their emission wavelengths are around 680 nm and 780nm in latex particles, respectively: Also preferred parent compounds arethe anthranylocyanines which have emissions around 850 to 900 nm. Thesethree classes of preferred parent compounds will collectively be called“phthalocyanine derivatives” and may or may not have an included metaland may or may not have axial ligands. Also, preferred parent compoundsinclude “hybrid phthalocyanine derivatives” which have 2 or moredifferent subunits of the 4 total subunits and may or may not have anincluded metal and may or may not have axial ligands. An example of ahybrid phthalocyanine derivative containing a metal and an axial ligandis illustrated in FIG. 9. The emission wavelengths for thephthalocyanine derivatives or the hybrid phthalocyanine derivatives areparticularly useful for quantifying fluorescence in biological samplesand tissues and for minimizing the background scatter intensity. Thoseskilled in the art can appreciate that phthalocyanine derivatives andhybrid phthalocyanine derivatives can be synthesized, for example, byderivatization of the phenyl, naphthyl or anthranyl rings with varioussubstitutes to yield different molecules. These variants are within thescope of the instant invention. Derivatives of tetraazaporphine are alsowithin the scope of the instant invention. The derivatization of thearomatic structure of phthalocyanine derivatives and hybridphthalocyanine derivatives can produce blue or red shifted excitation oremission wavelengths. The choice of the donor dye to excite thephthalocyanine or hybrid phthalocyanine derivatives is dependent onhaving a donor dye emission wavelength which corresponds to theappropriate range of absorbance wavelengths of the phthalocyanine orhybrid phthalocyanine derivative. FIG. 3 shows the absorbance spectra ofthe silicon dihydroxyphthalocyanine and silicondihydroxynaphthalocyanine in dimethylformamide. A potential range ofexcitation of the these acceptor dyes by the donor dye is betweenapproximately 550 nm and 670 nm and 600 nm and 760 nm, respectively. Oneskilled in the art will recognize that many dyes would be candidates forthe donor dye because of the wide useful range of wavelengths which canexcite the acceptor dyes. Indeed, the phthalocyanine derivative can bethe donor for the naphthalocyanine derivative. The choice of theacceptor dye should meet the criteria outlined above. Several examplesare described which illustrate the versatility of this novel approach.

If one wants to build an instrument with an excitation source which hasa maximum intensity at 480 nm and a detector which has a good quantumefficiency at 600 to 700 nm, the donor dye should be capable of beingexcited at 480 nm. Assuming that a phthalocyanine derivative is theacceptor dye for emission at 680 nm, the donor should then emit in therange of 550 to 670 nm.

Preferred classes of dyes for this application are styryl,phenylbutadienyl and phenylhexatrienyl dyes. Styryl dyes are those ofthe following formula:

and phenylbutadienyl dyes are of the formula:

and phenylhexatrienyl dyes are of the formula:

wherein R1, R2 and R3 can be the same or different and R1, R2 and R3 areH or alkylcarbon chains from 1-20 carbons, either saturated orunsaturated, and having 0-10 heteroatoms (N, O, S).

In general, these dye classes excite approximately between about 470 and530 nm and emit approximately between 600 and 780 nm (see MolecularProbes Handbook of Fluorescent Probes and Research Chemicals by RichardP. Haugland, 1992-1994, p. 156). A particularly preferred styryl dye isthe trans-4-[4-(dibutylamino)styryl]-1-methylpyridinium iodide (AldrichChemical Co.) which has its maximum absorbance at 486 nm indimethylformamide and its emission at 600 nm. One skilled in the artwill recognize that the substituents off the aniline nitrogen and thepyridium nitrogen of these classes of dyes can vary and that preferredsubstituents are those with hydrophobic groups to maintain waterinsolubility.

In another application, an instrument system is built which has a sourceof maximum intensity at 420 nm and a detector as described in the aboveexample. The dye system here can include the phthalocyanine acceptor;however, a different donor must be employed. A preferred donor for thisapplication is a meso-tetra-2-aminophenylporphine (Porphyrin Products,Inc., Logan Utah) which has a maximum absorbance for excitation at 418nm in dimethylsulfoxide and an emission around 655 nm. This porphyrinwill excite the phthalocyanine derivative in latex particles and the dyesystem will emit at 680 nm.

In a particularly preferred application, an instrument system is builtto perform immunoassays in neat blood or serum or in various biologicalspecimens. The excitation source is a light emitting diode (LED) orlaser diode which has a maximum intensity around 650 nm to avoidabsorption of the light by the blood or serum sample. The detector hasgood quantum efficiency at 700 to 800 nm so a preferred acceptor dye isa naphthalocyanine derivative which has an emission at approximately 780nm, an emission wavelength which is generally not in common with bloodor serum samples or biological specimens. A donor dye for thenaphthalocyanine acceptor should absorb at around 650 nm to coincidewith the source and emit between approximately 660 nm and 760 nm.Preferred classes of dyes for this donor application are thecarbocyanine dyes and the ethenyl-substituted dipyrrometheneborondifluoro dyes, as described in U.S. Pat. Nos. 5,187,288, 5,248,782 and5,274,113.

In yet another particularly preferred application, an instrument systemis built to perform immunoassays in neat blood, plasma or serum or invarious biological specimens. The excitation source is an LED or a laserdiode which has its maximum intensity around 670 nm to avoid absorptionof the light by the blood, plasma or serum sample. The detector has goodquantum efficiency at 700 to 800 nm so preferred acceptor dyes aresilicon[(diphthalocyanine) dinaphthalocyanine] ligands or anaphthalocyanine derivative which have an emissions at approximately 760nm and 780 nm, respectively, emission wavelengths which are generallynot in common with blood or serum samples or biological specimens. Adonor dye for the preferred acceptors should absorb at around 670 nm tocoincide with the source and emit between approximately 660 nm and 760nm. Preferred donor dyes are silicon phthalocyanine with axial ligands.

In yet another particularly preferred application, for immunoassays inneat blood or serum, the excitation source is around 790 nm and theemission wavelength is around 900 nm. A preferred dye for a single dyesystem is a silicon 1,6-octaethoxynaphthalocyaninebis(dimethylhexylvinylsilyloxide) which is excited at 790 nm and emitsat about 900 nm.

Preferred dyes for use as donor dyes for naphthalocyanines andnaphthalocyanine derivatives are, carbocyanines and ethenyl-substituteddipyrrometheneboron difluoro dyes, as described in U.S. Pat. Nos.5,187,288, 5,248,782 and 5,274,113 which have excitation wavelengths upto 790 nm and emission wavelengths between about 670 nm and 800 nm.

Preferred carbocyanine dyes, which generally excite between 500 and 750nm (see Molecular Probes Handbook) are of the general formula:

wherein n is 1 or 2; or 3; wherein R1 and R2 are S, N, or O; and whereinR3 and R4 are H or alkylcarbon chains of from 1-20 carbons, eithersaturated or unsaturated and having 0-10 heteroatoms (N, O, S).

Also preferred carbocyanine dyes are also of the general formula:

wherein n is 1 or 2; or 3; wherein R1-R6 are H or alkylcarbon chains offrom 1-20 carbons, either saturated or unsaturated and having 0-10heteroatoms (N, O, S).

Preferred donor dyes are also the ethenyl-substituteddipyrrometheneboron difluoro dyes, which generally excite above 500 mm(see Molecular Probes Handbook) and are of the general formula asdepicted in FIG. 4, wherein R1-R7 include substituents as described inU.S. Pat. Nos. 5,187,288, 5,248,782 and 5,274,113.

Particularly preferred donor dyes are1,1′-dihexyl-3,3,3′,3′-tetramethylindocarbocyanine iodide,1,1′-diethyl-3,3,3′,3′-tetramethylindodicarbocyanine iodide and(E,E)-3,5-bis-(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a,4a-diazo-5-indacene(from Molecular Probes Inc., Eugene, Oreg.) which have absorptionmaximums of 642 nm, and 645 nm and 650 nm and emission maximums of 674nm and 665 nm, and 670 mm, respectively, in dimethylformamide. Particlesincorporated with these particularly preferred dyes and anaphthalocyanine derivative will excite with a 650 nm source and emit atapproximately between 780 mm and 870 mm. One skilled in the art willrecognize that the excitation and emission spectra for any particulardye has a Gaussian form and therefore the excitation source does notneed to correspond exactly to the excitation maximum of the donor dye inorder to obtain an intense fluorescent signal. Likewise, the donoremission does not have to coincide with the highest absorption of theacceptor dye in order to achieve efficient energy transfer. One skilledin the art will also recognize that the substituents at and on the 1 and3 positions of the carbocyanines and the substituents at the R1 and R7positions of the dipyrrometheneboron difluoro dyes, and the conjugationbetween the ring structures can vary and these variations are alsouseful in tuning fluorescence spectra of the particles.

Also preferred emission wavelengths of fluorescent particles range fromabout 800 mm to 1000 mm. This near infra-red region is important becausethe scattering component of the light decreases substantially, thuslowering the background of the fluorescent measurement. In addition,biological samples do not absorb or fluoresce substantially in the 800mm-1000 mm range. Particulate materials in the samples, for example,lipoproteins in serum, particles in ground water, cellular debris inbiological samples and the like, can increase the background signalbecause of scattered light and the measurement of the scattered light isminimized in the 800-1000 nm range.

FIG. 5 illustrates the attenuation of the background signal as thewavelength of the measured light increases from 730 nm to 900 nm in animmunoassay device, as described in allowed application Ser. No.07/887,526 (which is herein incorporated by reference), containingeither neat human serum or no serum. This figure shows that thebackground signal decreases by a factor of 5 when measuring at 900 nm ascompared to 790 nm when the illumination source is a 1 milliwatt (“mW”)670 am laser diode. In addition, excitation of neat serum at 670 nm doesnot result in a significant measurable fluorescence between 730 nm and900 nm. Thus, for example, the signal to background ratio of themeasurement of fluorescence of a dye which emits at around 900 nm ascompared to a dye emitting at around 790 nm would be improved by afactor of 5. The signal to background ratio improves by a factor ofabout 30 when measuring emission at 780 nm as compared to 730 nm (seeFIG. 5). Preferred dyes, for example as described in J. Chem. Soc.Perkin Trans. 1, (1988), 2453-2458, which emit above 780 rim includederivatives of the naphthalocyanine and anthranylocyanine classes(FIG. 1) and the naphthalocyanine class is characterized by the generalformulae, as depicted in FIG. 6, where M is a metal such as Si, Ge, Al,Sn and Ti and the like, and where R is an axial ligand, alkyl or arylwith or without a silicon (preferred axial moieties are synthesized fromalkyl or aryl silyl chlorides), and where X is an electron donatinggroup or groups which can be the same or different, including, such asamino, hydroxyl, alkoxy, aryloxy, phenyl, alkyl and the like. Theelectron donating character of the X group or groups red-shifts theemission wavelength as compared to the general naphthalocyaninecompounds (FIG. 1).

For example, the compounds described in examples 26, 27 and 28 areillustrative of dyes which have emission wavelengths around 850 nm.These preferred dyes would yield an improved signal to background ratioas compared to dyes emitting at 780 nm (See FIG. 5). Electronwithdrawing groups can also be utilized for the X groups, such ashalogen, nitro, cyano, sulfate, carboxyl and carboxyalkyl and the like,which will blue shift the excitation or emission wavelengths. Preferreddonor dyes for this class of near infra-red emitting dyes are thosewhich have emission wavelengths which correlate to the absorbancecharacteristics of the acceptor dye. Preferred dyes for this applicationare the ethenyl-substituted dipyrrometheneboron difluoride dyes, asdescribed in U.S. Pat. Nos. 5,187,288, 5,248,782 and 5,274,113.

Preferred molar ratios of donor to acceptor dyes in the latex particlesgenerally range from about 20:1 to about 1:20 and particularly fromabout 1:1 to 6:1. The desired fluorescence intensity should be obtainedthrough experimentation using the principles taught herein, and byincorporating various ratios of donor to acceptor dyes into theparticles at various dye concentrations and measuring the fluorescenceemission of the particles.

The geometrical orientation of the dipoles of the donor and acceptordyes will affect the efficiency of energy transfer between them. Thedonor and acceptor dyes can be synthesized to form a compound of optimaldipole geometry, which, in solution, exhibits efficient fluorescenceenergy transfer (“FET”). The optimized FET compound then may beincorporated into particles. Phthalocyanine derivatives can be utilizedfor this application for the acceptor moiety, where the phthalocyaninederivative can be substituted with electron donating or withdrawinggroups (as described above) to accommodate the desired excitation andemission wavelength. For example, preferred naphthalocyanine compoundsfor this application are those as depicted in FIG. 7, where X ishydrogen or electron donating groups, such as amino, hydroxyl, alkoxy,aryloxy, phenyl, alkyl and the like and D is the donor dye covalentlyattached to the naphthalocyanine derivative at a distance which allowsfor energy transfer between the donor and acceptor.

By applying the teachings of this invention, all phthalocyanine ofhybrid phthalocyanine derivatives can function as donor or acceptormolecules. For example, a silicon ortho octaethoxy(phthalocyanine)derivative will emit at approximately 750 nm to 780 nm, similar to asilicon naphthalocyanine derivative. Generally, the distances betweendonor and acceptor are about 5 angstroms to 60 angstroms, and preferablyfrom 5 angstroms to 15 angstroms. In addition, each naphthalocyaninederivative can have 1-4 donor dyes attached, depending on the requiredapplication of the FET compound. Suitable donor dyes are those whichemit in the absorbance range of the acceptor dye. Example 29 describesthe synthesis of a fluorescein-silicon phthalocyanine FET compound.Table 1, item 56, shows the fluorescence characteristics of thiscompound in latex particles. One skilled in the art will appreciate thatwith the inventive teachings described herein, many FET compounds may besynthesized for many particular applications requiring specificexcitation and emission wavelengths.

Another approach to developing particles which exhibit desired andpredictable fluorescence properties in the high visible to near infraredspectrum is to synthesize unsymmetrical or hybrid phthalocyanines,naphthalocyanines or anthranylocyanines and their derivatives. As usedherein, the term “hybrid phthalocyanine derivatives” refers to allclasses of hybrid phthalocyanines, naphthalocyanines andanthranylocyanines and their derivatives, with or without metal andaxial ligands, including tetraazaporphines and their derivatives. Thenovel hybrid molecules described herein appear to exhibit intramolecularenergy transfer. The hybrid phthalocyanine derivatives can besynthesized from diiminoisoindoline or derivatives ofdiiminoisoindolines and incorporate a metal, for example, silicon, andelaboration with axial ligands or they can be synthesized fromdicarbonitrile derivatives of benzene, naphthalene or anthracenecompounds, respectively, for subsequent inclusion of various metals andelaboration with axial ligands. Hybrid molecules comprised ofderivatives of tetraazaporphines, as described in Inorg. Chem. (1994),33, 173 5-1740, are also within the scope of the hybrid phthalocyaninederivatives of the instant invention. A synthetic strategy for hybridphthalocyanine derivatives with 2 different subunits is described, forexample, in J. Am. Chem. Soc. (1990), 112, 9640-9641, Inorg. Chem.(1994), 33, 1735-1740, Chem. Letters, (1992), 763-766, Chem. Letters,(1992), 1567-1570 and Chem. Letters, (1992), 2031-2034. These referencesdescribe the synthesis of hybrid molecules with zinc metal or withoutmetal and without axial ligands. The character of the diiminoisoindolineand its derivatives will dictate the excitation and emissioncharacteristics of the molecule. Moreover, incorporation of dyes withaxial ligands, as taught herein, will result in particles which exhibitminimum quenching and maximum fluorescence intensity.

Axial ligands are also beneficial on water soluble compounds because theaxial ligands will minimize interaction of the hybrid molecule with, forexample, proteins, antibodies and nucleic acids, which may or may not becovalently coupled to the hybrid molecule. The axial ligand may itself,impart water solubility to the hybrid phthalocyanine derivative.

Examples of water soluble phthalocyanine derivatives are disclosed inExamples 92, 95-98, 108, 110, 114-124, and 126-128.

Novel hybrid phthalocyanine derivatives are described herein, whichcontain 3 or 4 different subunits, and allow for larger Stokes shifts.In these derivatives, excitation occurs with the subunit which has thehighest energy or the lowest wavelength absorption and the emissionoccurs in the lowest energy subunit.

The desired excitation and emission wavelengths of the hybridphthalocyanine derivative will determine the types of diiminoisoindolinederivative and dicarbonitrile derivative precursors which are used inthe synthesis of the hybrid phthalocyanines. The desired excitation andemission wavelengths are generally dictated by the sample, the type offluorescent measurement and the instrument. Various combinations ofdiiminoisoindoline derivative and dicarbonitrile derivative precursorsalso may be combined to form a hybrid phthalocyanine derivative whichmay have a red shifted or blue shifted excitation and/or emissionwavelength pattern.

In general, electron donating substituents on the diiminoisoindoline ordicarbonitrile precursors, particularly situated at the ortho positions(that is, ortho to the tetraazaporphine structure as indicated in FIG. 6for the X substituents) of the phthalocyanine structure, such as amino,hydroxyl, alkoxy, aryloxy, phenyl, alkyl and the like, will red shiftthe excitation and/or emission wavelengths. Conversely, electronwithdrawing substituents, also particularly at the ortho positions, suchas halogen, nitro, cyano, sulfate, carboxyl and carboxyalkyl and thelike, will blue shift the excitation or emission wavelengths. Inaddition, positions on the subunits other than the ortho positions canaffect the excitation and emission characteristics of the hybridphthalocyanine derivative. The choice of either diiminoisoindoline ordicarbonitrile precursors for the synthesis of the hybrid phthalocyaninederivatives is related to the desired presence or absence of metal andthe type of metal in the hybrid molecule. For example, when using thediiminoisoindoline precursors in the synthesis, a silicon metal can beincorporated during the tetramerization reaction to form thephthalocyanine derivative structure. The silicon can be further modifiedto a silicon dihydroxy phthalocyanine derivative molecule so that axialligands can be elaborated with, for example, various silyl chloridereagents. The importance of axial ligands in reducing quenching andmaximizing fluorescence intensity is evident for bothphthalocyanine/naphthalocyanine molecules and the hybrid phthalocyaninederivatives (see example 65).

The axial ligands are also useful for further elaboration of themolecules, for example, for attaching another fluorescent molecule, forattaching to a ligand, protein, polypeptide or nucleic acid or forchanging the charge of the molecule using sulfate, carboxylic acid oramino substituents which can affect solubility of the molecule. In thecase of using axial ligands to attach the water soluble dye to ligands,proteins, polypeptides or nucleic acids, a mono- or bis-substitutedmetal can be utilized. The mono-substituted metal in the dye, however,yields only one axial ligand onto which the chemistry of attachment ismade. The other face of the dye, after attachment to a ligand, protein,polypeptide or nucleic acid, which has no axial ligand, may interactwith neighboring molecules (proteins, polypeptides, nucleic acids andthe like) and result in quenching of fluorescence. The bis-substituteddye can minimize potential interactions between neighboring moleculeswhen one axial ligand is used for attachment and the other isunattached. In this case, the unattached axial ligand can be synthesizedsuch that the terminal atom of the unattached axial ligand imparts watersolubility to the molecule, for example, a sulfate, carboxyl, or anamino derivative, such that interactions between neighboring moleculesis minimized. In the case of utilizing water soluble hybridphthalocyanine derivatives, for example, for competitive immunoassays,the ligand analogue of the target ligand which is being measured, can beattached to the dye through the axial ligand(s). The axial ligands ofthe water soluble phthalocyanine and hybrid phthalocyanine derivativescan also contain functional groups, for example, amines, carboxylicacids and esters, alkyl halides, thiols, thio ester and the like forattachment of ligands, proteins, polypeptides and nucleic acids. Theaxial ligands can also impart water solubility on the phthalocyanine andhybrid phthalocyanine derivatives when the axial ligand is comprised ofpoly(ethylene oxide). The carboxylic acid ester or the thioester groupson the axial ligands can be hydrolyzed in dilute base to the carboxylicacid and thiol groups, respectively. The chemical reactions to attachthe axial ligands to ligands and ligand analogues, proteins,polypeptides and nucleic acids should be compatible with the functionalgroups of the compounds or macromolecules. For example, an amine on theaxial ligand of the dye can be reacted with a compound or macromoleculecontaining a carboxylic acid or an alkyl halide, an alkyl halide on theaxial ligand of the dye can be reacted with an amine or a thiol on thecompound or macromolecule, a thiol on the axial ligand of the dye can bereacted with an alkyl halide or a maleimide group on the compound ormacromolecule. Thus, compounds, such as ligands, ligand analogues andmacromolecules, such as nucleic acids, polypeptides and antibodies canbe reacted specifically to the dye by reaction with functional groups onthe dye.

In general, phthalocyanine and hybrid phthalocyanine derivatives can bemade water soluble by sulfonating the compounds using, for example,sulfuric acid or chlorosulfonic acid (see Gilbert, “Sulfonation andRelated Reactions”, Interscience, New York, 1965; Cerfontain,“Mechanistic Aspects in Aromatic Sulfonation and Desulfonation”,Interscience, New York, 1968, Int. J Sulfur Chem. C6, 123-136 (1971)).The sulfonation of the aromatic ring structure of the dye molecules canoccur at various carbons of the ring. Added water solubility of the dyemolecules can be achieved using axial ligands comprised of poly(ethylene oxide).

When using the dicarbonitrile precursors, the phthalocyanine derivativeis synthesized without metal, but various metals can subsequently beincluded, for example, Ge, Al, Sn, Ti and the like. These metals canalso be elaborated with axial ligand(s), depending on the valence of themetal.

The fluorescence quenching character of the hybrid phthalocyaninederivatives in particles are particularly preferred over thephthalocyanine derivatives. Example 66 is a typical example ofcomparison of the quenching characteristics in latex particles ofsilicon 2,3-naphthalocyanine-bis(dimethylhexylvinylsilyloxide) andsilicon-[di(1,6-diphenylnaphthalocyanine)]-diphthalocyanine-bis-(dimethylhexylvinylsilyloxide).The hybrid phthalocyanine derivative has essentially no quenching ascompared to up to 50% quenching of the naphthalocyanine derivative forthe various dye loading concentrations listed in the table. Thefluorescence intensity of latex containing the hybrid phthalocyaninederivative are much greater than the phthalocyanine derivative. Thisillustrates the special properties of the hybrid phthalocyaninederivatives.

The hybrid phthalocyanine derivatives are also very good acceptors whenusing phthalocyanine derivatives as donors. This is shown in table 6 ofexample 67. When the phthalocyanine derivative is the donor and thehybrid phthalocyanine derivative is the acceptor (dye system 3), thefluorescence intensity of the particles is about 145% higher than whenthe same phthalocyanine derivative is the donor and a naphthalocyaninederivative is the acceptor (dye system 2). These results show thespecial properties of the hybrid phthalocyanine derivative in particlesexhibiting fluorescence energy transfer.

The hybrid phthalocyanine derivative also acts as an intermediate donorcompound. Table 6 of Example 67 shows that the fluorescence intensity ofa naphthalocyanine acceptor in a particle prepared in 70%tetrahydrofuran (dye system 4) is increased about 65% when aphthalocyanine donor excites a hybrid phthalocyanine compound ascompared to the phthalocyanine donor directly exciting thenaphthalocyanine acceptor (dye system 2). These results furtherillustrate the special properties of the hybrid phthalocyaninederivatives in latex particles exhibiting fluorescence energy transfer.

The results of Table 6 of Example 67 also show the ability ofphthalocyanine derivatives with axial ligands to exhibit singlet-singletenergy transfer to other phthalocyanine or hybrid phthalocyaninederivatives with axial ligands. That is, it is apparent from Example 65and Table 4, that axial ligands reduce the quenching of the dyes andenhance the fluorescence of the particles. Other experiments (seeExample 15, Tables 1 and 2) also support this observation. Thus, axialligands minimize quenching by preventing the close contact of the ringstructures. One would then expect that phthalocyanine or hybridphthalocyanine derivatives with axial ligands would not be spacedsufficiently close to function efficiently as energy transfer donor andacceptor pairs because the molecules are spaced apart by the axialligands. However, nearly 100% efficiency of energy transfer and highfluorescence intensities are observed in particles when phthalocyanineor hybrid phthalocyanine derivatives with axial ligands are donors andphthalocyanine or hybrid phthalocyanine derivatives are acceptors.

The tetramerization reactions of the diiminoisoindoline ordicarbonitrile precursors to form the hybrid phthalocyanine derivativescan be directed so that opposing subunits can be the same. This isaccomplished, for example, with the use of bulky substituents on theprecursors so that in the tetramerization reaction, like subunits withbulky substituents cannot be adjacent because of steric considerations.Bulky phenyl substituents have been used on dicarbonitrile precursors todirect the precursors tetramerization to be opposing subunits asdescribed Inorg. Chem. (1994), 33, 1735-1740, Chemistry Letters (1992),2031-2034 and Chemistry Letters (1992), 1567-1570.

Preferred hybrid phthalocyanine derivatives have similar opposingsubunits so that two different subunits comprise the structure.Particularly preferred hybrid phthalocyanine derivatives have similaropposing subunits on one axis and different opposing subunits on theother axis. The nature of the particularly preferred molecules is thatred or blue shifted excitation or emission wavelengths and a longerStokes shift can result because of the selection of the precursormolecules for the tetramerization reaction. For particularly preferredhybrid phthalocyanine derivatives, for example, the “donor”diphenyldiiminoisoindoline or the diiminoisoindoline precursors wouldcontribute to 650 nm absorbance of the hybrid molecule, and thereby tothe excitation of the hybrid molecule. The diphenylphenyldiiminoisoindoline or the phenyldiiminoisoindoline precursorswould act as an “electron transfer subunit” to the “acceptor subunit”,which would be a dialcoxy or aryloxy phenyldiiminoisoindolineprecursors, so that emission is dictated at the lowest energy by theacceptor subunit at about 850 nm. The nature of the “electron transfersubunit” is important because it is not desirable for this subunit toemit because then the desired emission of the acceptor subunit will nottake place. Thus, the highest occupied molecular orbital (HOMO) andlowest unoccupied molecular orbital (LUMO) character of the electrontransfer subunit should be designed with reference to the donor andacceptor subunit molecules. The relationship of the energies of the HOMOand LUMO as they relate to excitation and emission are taught by Pariseret al., J. Chem. Phys. (1953), 21, 767-776, by Pople, Trans. FaradaySoc. (1953), 49, 1375-1385, by McHugh et al., Theoret. Chim. Acta(Berlin) (1972), 24, 346-370 and by Kobayashi et al, Inorg. Chem.(1994), 33, 1735-1740, Chemistry Letters (1992), 2031-2041, Konami etal., Molecular Physics (1993), 80, 153-160.

Another application requires the hybrid molecule to have two excitationwavelengths, one at approximately 650 nm and another at about 680 nmwith emission for both excitations at about 760 nm. Thus, the precursorsresponsible for the excitation could be a diiminoisoindoline for the 650mm and a tetrafluorodiiminoisoindoline for the 680 nm excitations. Theemitting subunit, which can also be used to direct the tetramerizationreaction so that the emitting subunits are opposed in the molecule, canbe a diphenyl phenyldiiminoisoindoline. The excitation and emissionwavelengths of the resulting hybrid phthalocyanine derivative are thusgenerally representative of the individual diiminoisoindolineprecursors.

Yet another application requires excitation at about 650 nm and emissionat about 750 nm. The precursors responsible for excitation and emissioncould be diiminoisoindoline and diphenyl phenyldiiminoisoindoline,respectively. The latter precursor also acts to direct the emittingsubunits to be opposed.

In another application, a large extinction coefficient at the excitationwavelength is desired for excitation at about 650 nm. The emissionwavelength should be at about 850 nm. The precursors responsible forexcitation could be a diphenyldiiminoisoindoline, which would directthese subunits to be opposed and thereby two subunits would contributeto provide the desired extinction coefficient. Aphenyldiiminoisoindoline derivative precursor could act as an electrontransfer subunit and an alkoxy-phenyldiiminoisoindoline precursor couldbe the acceptor with a characteristic emission at about 850 nm.

In another application, two emission wavelengths are desired from acompound which is excited at a single wavelength. The desired excitationis around 650 nm and the emission should be around 760 nm and 810 nm.The precursor responsible for excitation could be atetrafluorodiiminoisoindoline or atetrafluorobenzene-1,2-dicarbothtrile. The precursor responsible foremission could be a dibutoxy-phenyldiiminoisoindoline or a3,4-dibutoxy-naphthalene-1,2-dicarbonitrile, respectively.

Incorporation of Dyes into Particles

The resulting compounds are then incorporated into particles to yieldparticles which exhibit excitation wavelengths above about 600 nm andemission wavelengths above about 650 nm. One skilled in the art willalso appreciate that water soluble hybrid phthalocyanine derivatives arevaluable for coupling to proteins, polypeptides, nucleosides, nucleicacids and the like, for detecting their presence in biological fluids orfor performing DNA probe or immunoassays.

Preferred particle sizes range from about 0.1 nm to 5000 nm andpreferably from about 1 nm to 1000 nm. The choice of particle sizeshould be related to the specific function for the label. The particlesize may vary for a particular application. For example, in animmunoassay, if the label requires a more intense fluorescence formeasuring very low concentrations of analytes, then one would employlarger particles because larger particles can incorporate more dyemolecules. The small particle sizes (0.1-1 nm) may be employed influorescence polarization assays, as described for example, in U.S. Pat.Nos. 4,420,568, 4,476,229 and 4,510,251, in in vitro visualization ofcellular components or in in vivo imaging techniques.

The resulting fluorescent dye particles which exhibit the appropriateexcitation and emission characteristics are further adsorbed orchemically reacted with various nucleic acids, nucleotides, proteins orpeptides and the like which are required for a specific purpose. Theadsorption of macromolecules to particles, particularly latex particlesis well known to those skilled in the art and generally involvesadsorption of the macromolecule at a temperature between 5° C. and 50°C. and at a pH which is below the pI of the molecule.

Use of Incorporated Dye Particles in Assays

Fluorescent particles exhibiting fluorescence energy transfer can beadsorbed with either antibodies for use in non-competitive immunoassaysor ligand analogues for use in competitive immunoassays in reactionmixtures of the assays. In the case of non-competitive assays, thereaction mixture would include at least one target ligand and at leastone class of fluorescent particles having bound thereto at least onereceptor specific for target ligand, forming an antibody (fluorescent)conjugate. In the case of competitive assays, the reaction mixture willinclude at least one target ligand, at least one receptor specific tothe target ligand, and at least one class of fluorescent particles,having bound thereto at least one ligand analogue, forming a ligandanalogue (fluorescent) conjugate. The antibody conjugates bound totarget ligands in the non-competitive reaction mixture and the ligandanalogue conjugates not bound by receptors specific to the targetligands in the competitive reaction mixture can be bound to a solidphase consisting of receptors specific to another epitope of the targetligand of the target ligand antibody conjugate complexes and ofreceptors specific to ligand analogues of the ligand analogueconjugates, respectively. The fluorescent conjugates unbound by thesolid phase are removed and the fluorescence of the bound conjugates ismeasured. The measured fluorescence is related to the target ligandconcentration. The various reagents described above can also be attachedcovalently to the latex particles. For example, antibodies or ligandanalogues can be attached through amine or carboxylic acids tocarboxylic acids or amines on the surface of the particles,respectively, to form stable amide linkages.

In the case of quantifying nucleic acids in samples, the novel compoundsdescribed in the instant invention are useful because of theirbrightness and because of the near infrared emission characteristics. Ingeneral, in designing an assay for a nucleic acid, one selects a probemolecule which is complementary to the nucleic acid to be quantified.The probe molecule is then labeled, generally covalently, with a signalgenerator. The signal generator can be a water soluble phthalocyaninederivative or hybrid phthalocyanine derivative or a particle with theappropriate dye system, which may exhibit fluorescence energy transferor hybrid phthalocyanine derivatives or combinations of these compounds.The labeled probe molecule is then introduced into a biological samplesuspected of containing the target nucleic acid, and the labeled probesequence assembles with the target nucleic acid. The labeledprobe/target nucleic acid can then be immobilized onto a surface whichhas immobilized another nucleic acid which is also complementary to thetarget nucleic acid. Conversely, the biological sample can be introducedto a surface which has immobilized a complementary nucleic acid forimmobilization of the target nucleic acid. The labeled probe can then beintroduced to the system for binding to the immobilized target molecule.The excess labeled probe is then washed away and the resultantfluorescent intensity is correlated with fluorescence intensity from astandard curve to arrive at a concentration of the nucleic acid in thesample.

Use of Water Soluble Hybrid Phthalocyanine Derivatives in Assays

Water soluble hybrid phthalocyanine derivatives can be attached toantibodies for use in non-competitive immunoassays or ligand analoguesfor use in competitive immunoassays in reaction mixtures of the assays.In the case of non-competitive assays, the reaction mixture wouldinclude at least one target ligand and at least one water soluble hybridphthalocyanine derivative having attached thereto at least one receptorspecific for target ligand, forming an antibody (fluorescent) conjugate.In the case of competitive assays, the reaction mixture will include atleast one target ligand, at least one receptor specific to the targetligand, and at least one water soluble hybrid phthalocyanine derivativehaving attached thereto at least one ligand analogue, forming a ligandanalogue (fluorescent) conjugate. In addition, in certain embodiments,the antibody conjugates and ligand analogue conjugates can be utilizedas non-fluorescent labels. The non-fluorescent labels would be used inapplications where only a color response, measured by reflectance in anassay device, is necessary.

The fluorescent conjugates of water soluble hybrid phthalocyaninederivatives, which are smaller in molecular weight than the fluorescentparticles described herein, will diffuse faster in solution and resultin binding reactions which have faster kinetics. Fast kinetics of thebinding reactions in assays are preferred because the assays will reachequilibrium binding in a shorter time, and in turn, assay results can beobtained in a shorter time. The antibody conjugates bound to targetligands in the non-competitive reaction mixture and the ligand analogueconjugates not bound by receptors specific to the target ligands in thecompetitive reaction mixture can be bound to a solid phase consisting ofreceptors specific to another epitope of the target ligand of the targetligand-antibody conjugate complexes and of receptors specific to ligandanalogues of the ligand analogue conjugates, respectively. Thefluorescent conjugates unbound by the solid phase are removed and thefluorescence (or color) of the bound conjugates is measured. Themeasured fluorescence (or color) is related to the target ligandconcentration.

In the case of quantifying nucleic acids in samples, the novel compoundsdescribed in the instant invention are useful because of theirbrightness and because of the near infrared emission characteristics. Ingeneral, in designing an assay for a nucleic acid, one selects a probemolecule which is complementary to the nucleic acid to be quantified.The probe molecule is then labeled, generally covalently, with a signalgenerator. The signal generator can be a water soluble phthalocyaninederivative or hybrid phthalocyanine derivative. The labeled probemolecule is then introduced into a biological sample suspected ofcontaining the target nucleic acid, and the labeled probe sequenceassembles with the target nucleic acid. The labeled probe/target nucleicacid can then be immobilized onto a surface which has immobilizedanother nucleic acid which is also complementary to the target nucleicacid. Conversely, the biological sample can be introduced to a surfacewhich has immobilized a complementary nucleic acid for immobilization ofthe target nucleic acid. The labeled probe can then be introduced to thesystem for binding to the immobilized target molecule. The excesslabeled probe is then washed away and the resultant fluorescentintensity is correlated with fluorescence intensity from a standardcurve to arrive at a concentration of the nucleic acid in the sample.

Those skilled in the art will recognize that many variations ofimmunoassays and nucleic acid assays can be performed and the inventiveteachings in the instant invention for the use of novel dye systems canbe used to develop novel adaptations to existing technologies.

Those skilled in the art will appreciate that the novel fluorescentparticles and dyes described herein have many uses in immunoassays,fluorescence microscopy, in vivo imaging, in vitro cancer therapy,nucleic acid assays, cell sorters and the like.

Experimental Section

Fluorescence measurements referred to in the following Examples wereperformed on a Perkin-Elmer model LS 50B Luminescence Spectrometer fordyes emitting up to around 780 nm. In some instances, as indicated inTable 1 by describing the Intensity in terms of nanoamps (nA), dyesemitting above 800 nm were measured according to Example 18. Thefluorescence intensities are not corrected. Absorbance measurements wereperformed on a Hewlett Packard 8452A Diode Array Spectrophotometer.

EXAMPLE 1 Synthesis of Silicon Phthalocyanine Dihydroxide SiPc(OH)₂

A suspension of silicon phthalocyanine dichloride (1.83 g, 3.0 mmol) inpyridine (50 ml) and water (50 ml) was refluxed with stirring on an oilbath at 120° C. for 18 hours. After cooling the dark blue solid productwas filtered and the residue was washed with water (10 ml), acetone (5ml) and then dried under vacuum to afford 1.71 g of the title compound.

EXAMPLE 2 Synthesis of Silicon Phthalocyanine bis(trihexylsilyloxide)(hereinafter sometimes referred to as PcSi trihexyl)

A suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol) inanhydrous pyridine (11 ml) containing chlorotrihexylsilane (733 μL, 2.0mmol) was refluxed on an oil bath at 130° C. for 5 hours. The resultingpurple solution was allowed to cool and was evaporated. The resultingslurry was treated with ice-cold hexane (2 ml) and the dark blue solidproduct was filtered, washed with ice-cold hexane (2 ml) and was driedunder vacuum to yield 249 mg of crude product. The crude product inchloroform was purified on an Alumina column (Activity 1) equilibratedin hexane and the product was eluted with hexane/toluene (2/1, v/v) as abright blue band. The solvent containing the product was evaporated toyield 69 mg of the title compound with a melting point of (mp) 171° C.(literature mp is 175° C.).

EXAMPLE 3 Synthesis of Silicon Phthalocyaninebis[(10-carbomethoxydecyl)dimethylsilyloxidel (Hereinafter sometimesreferred to as PcSi methyl ester)

To a suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol)in anhydrous pyridine (11 ml) was added(10-carbomethoxydecyl)dimethylchlorosilane (586 mg, 2 mmol) and themixture was refluxed with stirring on an oil bath at 130° C. for 5hours. The dark blue solution was allowed to cool and the solvent wasevaporated. The residue was purified on a Silica gel 60 Å columnequilibrated in hexane and the product eluted slowly as a blue band withtoluene. The toluene fraction containing product was evaporated, hexane(10 ml) was added to the residue and the blue product was filtered,washed with hexane and dried to afford 105 mg of the title compound.

EXAMPLE 4 Synthesis of Silicon Phthalocyaninebis(dimethylvinylsilyloxide) (Hereinafter sometimes referred to as PcSivinyl)

To a suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol)in anhydrous pyridine (11 ml) was added chlorodimethylvinylsilane (276μL, 2.0 mmol) and the mixture was refluxed with stirring on an oil bathat 130° C. for 5 hours. The dark solution was allowed to cool and wasevaporated. The residue was purified on a Silica gel 60 Å columnequilibrated in hexane and the product was eluted with toluene as a blueband. The eluate containing product was evaporated, the residue treatedwith hexane and the dark blue solid product was filtered, washed withhexane and was dried under vacuum to afford 7.5 mg of the titlecompound.

EXAMPLE 5 Synthesis of Silicon Phthalocyaninebis[(3-cyanopropyl)dimethylsilyloxidel (Hereinafter sometimes referredto as PcSi cyano)

To a suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol)in anhydrous pyridine (11 ml) was addedchloro(3-cyanopropyl)-dimethylsilane (328 μL, 2.0 mmol) and the mixturewas refluxed with stirring on an oil bath at 130° C. for 5 hours. Thepurple solution was allowed to cool and was evaporated. The residue waspurified on a Silica gel 60 Å column equilibrated in hexane. The columnwas washed with toluene and the product was eluted withtoluene/isopropyl alcohol (90/10, v/v) as a bright blue band. The eluatecontaining product was evaporated under vacuum to afford 101 mg of thetitle compound with a mp>260° C.

EXAMPLE 6 Synthesis of Silicon Phthalocyaninebis(dimethylpentafluoro-phenylsilyloxide) (Hereinafter sometimesreferred to as PcSi pentafluoro)

To a suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol)in anhydrous pyridine (11 ml) was addedchlorodimethylpentafluorophenylsilane (376 μL, 2.0 mmol) and the mixturewas refluxed with stirring on an oil bath at 130° C. for 5 hours. Thedark green solution was allowed to cool and was evaporated. The residuewas purified on a Silica gel 60 Å column equilibrated in hexane. Theproduct was eluted with toluene as a dark blue band. The eluatecontaining the product was evaporated, the residue was treated withhexane (10 ml) and the dark blue solid product was filtered, washed withhexane and was dried under vacuum to afford 73 mg of the title compound.

EXAMPLE 7 Synthesis of Silicon 2,3-Naphthalocyanine Dihydroxide(Hereinafter sometimes referred to as NaPcSi hydroxide)

A suspension of silicon 2,3-naphthalocyanine dichloride (280 mg, 0.34mmol) in pyridine (10 ml) and water (10 ml) was refluxed with stirringon an oil bath at 130° C. for 24 hours. After cooling to roomtemperature, the dark green solid product was filtered and, the residuewas washed, successively, with water (5 ml) and acetone (2 ml). Theproduct was dried under vacuum to afford 217 mg of the title compound.

EXAMPLE 8 Synthesis of Silicon 2,3-Naphthalocyaninebis(dimethylvinylsilyloxide) (Hereinafter sometimes referred to asNaPcSi vinyl)

To a suspension of silicon 2,3-naphthalocyanine dihydroxide (87 mg, 0.11mmol) in anhydrous dimethylformamide (1 ml) was addedchlorodimethylvinylsilane (0.042 ml, 0.3 mmol), followed by imidazole(14 mg, 0.2 mmol). The mixture was stirred under argon at roomtemperature for 24 hours. The solvent was evaporated and the residue waspurified on a Silica gel 60 Å column which was equilibrated in hexane.The product was eluted with toluene as a green band. The toluenefraction containing the product was evaporated and the residue wastreated with hexane. The dark green solid was filtered, washed withhexane and was dried under vacuum to afford 26 mg of the title compound.

EXAMPLE 9 Synthesis of Silicon 2,3-Naphthalocyaninebis(dimethylpentafluorophenylsilyloxide) (Hereinafter sometimes referredto as NaPcSi pentafluoro)

To a suspension of silicon 2,3-naphthalocyanine dihydroxide (87 mg, 0.11mmol) in anhydrous pyridine (5 ml) was addedchlorodimethylpentafluorophenylsilane (0.188 ml, 1 mmol). The mixturewas refluxed with stirring on an oil bath at 130° C. for 5 hours. Aftercooling, the solvent was evaporated and the residue was purified on aSilica gel 60 Å column which was equilibrated in hexane. The product waseluted with toluene as a green band. The toluene fraction containing theproduct was evaporated and the residue was treated with hexane. The darkgreen solid was filtered, washed with hexane and was dried under vacuumto afford 23 mg of the title compound.

EXAMPLE 10 General Preferred Procedures for the Preparation ofDye-Loaded Latex Particles of Varying Sizes

The various dyes were loaded into latex particles of varying sizesaccording to the general procedures outlined below. The proceduresdescribed involve swelling latex particles with aqueous solutions ofeither tetrahydrofuran or dimethylformamide prior to addition of the dyesolutions. Latex particle sizes used range from 67 nm to 783 nm and oneskilled in the art recognizes that smaller and larger particles can beused. Tables 1 and 2 of Example 15 below show the aqueous organicsolvent system and the optimum dye concentration which were used for theloading into particles for each dye pair or for hybrid phthalocyaninederivatives, respectively, of a selected number of dyes. One skilled inthe art recognizes that many changes can be made to these procedures toprepare particles with different degrees of fluorescence intensities andquenching by loading higher or lower amounts of dye in the particles andalso by changing the ratios of each dye pair to the other. One skilledin the art also recognizes that similar techniques are useful forincorporation of dyes into latex particles, for example, as described inU.S. Pat. Nos. 4,199,363 and 4,368,258.

Surfactant-free polystyrene sulfate latex particles in sizes rangingfrom 67 nm to 783 nm and carboxyl-modified latex (“CML”) particlesranging from 200 nm to 400 nm particles were obtained throughInterfacial Dynamics Corp. Inc., Portland Oreg.

Method 1. Utilizing Tetrahydrofuran

a. 20% Tetrahydrofuran

Tetrahydrofuran (0.09 ml) was added, dropwise over a 5 minute period, toa stirring solution of 0.5 ml of 2.0% solids of latex particles at roomtemperature. The latex suspension was stirred at room temperature for anadditional 30 minutes to swell the latex. The dye solution (0.01 ml),which consists of one or more dyes at an appropriate concentration intetrahydrofuran, was added dropwise over 5 minutes to the stirred latexsolution, to give the loading dye concentration (in a 0.6 ml volume) asindicated in Table 1. The latex-dye solution was stirred at roomtemperature for 30 minutes in the dark. The latex solution was thentransferred to dialysis tubing (Spectra-por, 12-14,000 molecular weightcutoff, Spectrum, Houston, Tex.) and the dye-latex solutions weredialyzed against water for 12-15 hours at 4° C. The dye-latex solutionwas removed from dialysis and the % solids of the solution wascalculated from the final volume after dialysis and the starting solidsconcentration.

b. 50% Tetrahydrofuran

Tetrahydrofuran (0.20 ml) was added, dropwise over a 5 minute period, toa stirring solution of 0.24 ml of 4.1% solids of latex particles at roomtemperature. The latex suspension was stirred at room temperature for anadditional 30 minutes to swell the latex. The dye solution (0.06 ml),which consists of one or more dyes at an appropriate concentration intetrahydrofuran, was added dropwise over 5 minutes to the stirred latexsolution, to give the loading dye concentration (in a 0.5 ml volume) asindicated in Table 1. The latex-dye solution was stirred at roomtemperature for 30 minutes in the dark. The latex solution was thendialyzed and analyzed according to the procedures outlined in the 20%tetrahydrofuran method.

c. 70% Tetrahydrofuran

Tetrahydrofuran (0.29 ml) was added, dropwise over a 5 minute period, toa stirring solution of 0.15 ml of 6.7% solids of latex particles at roomtemperature. The latex suspension was stirred at room temperature for anadditional 30 minutes to swell the latex. The dye solution (0.06 ml),which consists of one or more dyes at an appropriate concentration intetrahydrofuran, was added dropwise over 5 minutes to the stirred latexsolution, to give the loading dye concentration (in a 0.5 ml volume) asindicated in Table 1. The latex-dye solution was stirred at roomtemperature for 30 minutes in the dark. The latex solution was thendialyzed and analyzed according to the procedures outlined in the 20%tetrahydrofuran method.

Method 2. Utilizing Dimethylformamide

a. 50% Dimethylformamide

Dimethylformamide (0.20 ml) was added, dropwise over a 5 minute period,to a stirring solution of 0.24 ml of 4.1% solids of latex particles atroom temperature. The latex suspension was stirred at room temperaturefor an additional 30 minutes to swell the latex. The dye solution (0.06ml), which consists of one or more dyes at an appropriate concentrationin dimethylformamide, was added dropwise over 5 minutes to the stirredlatex solution, to give the loading dye concentration (in a 0.5 mlvolume) as indicated in Table 1. The latex-dye solution was stirred atroom temperature for 30 minutes in the dark. The latex solution was thentransferred to dialysis tubing (Spectra-por, 12-14,000 molecular weightcutoff; Spectrum, Houston, Tex.) and the dye-latex solution was dialyzedagainst water for 12-15 hours at 4° C. The dye-latex solution wasremoved from dialysis and the % solids of the solution was calculatedfrom the final volume after dialysis and the starting solidsconcentration.

b. 70% Dimethylformamide

Dimethylformamide (0.29 ml) was added, dropwise over a 5 minute period,to a stirring solution of 0.15 ml of 6.7% solids of latex particles atroom temperature. The latex suspension was stirred at room temperaturefor an additional 30 minutes to swell the latex. The dye solution (0.06ml), which consists of one or more dyes at an appropriate concentrationin dimethylformamide, was added dropwise over 5 minutes to the stirredlatex solution, to give the loading dye concentration (in a 0.5 mlvolume) as indicated in Table 1. The latex-dye solution was stirred atroom temperature for 30 minutes in the dark. The latex solution was thendialyzed and analyzed according to the procedures outlined in the 50%dimethylformamide method.

EXAMPLE 11 Effect of Varying Dye Loading Concentration on FluorescenceIntensity and Optimization of Fluorescence Intensity Latex Particles

The incorporation of dye into latex particles must be optimized in orderto achieve the maximum fluorescence intensity and to minimize the degreeof fluorescence quenching of the dye molecules. Fluorescence quenchingcan be significant because of the close proximity of the dye moleculesin the particles. The PcSi vinyl was incorporated into 67 nm latexparticles (polystyrene sulfate from Interfacial Dynamics Corp. (DC),Inc., Portland, Oreg.) using method 1 (example 10) at variousconcentrations as indicated in the table below. The dye latex particleswere diluted to 0.0019% solids in either water or tetrahydrofuran foreach dye concentration. The solutions were excited at 350 nm and theemission at 680 nm was measured. The percent quenching in the particlesis: (1 fluorescence intensity in water divided by the intensity in theorganic solvent])×100. The table below shows the fluorescenceintensities as a function of dye loading concentrations and quenchingfor each condition. Loading Dye Concentration (mg/ml) Intensity (680 nm)Quenching(%) 0.01 420 41 0.025 489 65 0.05 492 73 0.075 401 76 0.1 33883 0.15 197 87 0.3 91 90 0.9 34 96

These results show that an optimum loading dye concentration gives thehighest fluorescence intensities and the lowest quenching. In this case,a dye concentration of between 0.025 and 0.05 mg/ml in the loadingsolution gives the best intensity and the least quenching. Less dye than0.025 mg/ml gives less intensity and less quenching because the spacingof the dyes begins to significantly increase and more dye than 0.05mg/ml gives less intensity and more quenching because of the increasedcloseness of the dyes in the particles. This type of experimentillustrates the procedure for optimization of fluorescence intensity andfor minimizing quenching.

EXAMPLE 12 Verification of Fluorescence Energy Transfer in LatexParticles

The latex particles which were incorporated with various dyes for energytransfer were diluted to 0.06% to 0.001% solids in water and eithertetrahydrofuran or dimethylformamide and the solutions of equal solidsconcentrations were excited at wavelengths which corresponded to theapproximate excitation maximum of the donor dye. The particles werediluted into organic solvents in order to liberate the dyes from thelatex, and therefore, disrupt any energy transfer process between thedyes in the particles. The fluorescence of the solutions in water andorganic solvent at the emission maximum of the acceptor dye or dyes wererecorded and compared. Fluorescence energy transfer was defined assignificant when the emission intensity of the acceptor was at least5-fold higher in water than in the organic solvent.

EXAMPLE 13 Effect of Varying Donor Dye Concentration with Respect toAcceptor Dye Concentration in Latex Particles on the FluorescenceIntensity of the Particles

Meso-tetra-2-dimethylaminophenyl porphyrin was made as follows. To astirring solution of meso-tetra-2-aminophenyl porphyrin (100 mg, 0.15mmol) and 37% aqueous formaldehyde (500 μL, 6.0 mmol) in tetrahydrofuran(2.5 ml was added sodium cyanoborohydride (114 mg, 1.8 mmol). Themixture was then treated with a glacial acetic acid (60 μL) over 10minutes and stirred at room temperature for 3 hours. More glacial aceticacid (60 μL) was added and the mixture stirred a further 1 hour at roomtemperature. The mixture was evaporated and the residue was purified ona Silica gel 60 Å column which was equilibrated in toluene. The productwas eluted with toluene/1% isopropanol as a dark brown band. Thefraction containing the product was evaporated and the ink-blue solidresidue dried under vacuum to afford 85 mg of the title compound.

Meso-tetra-2-dimethylaminophenyl porphyrin (Tdap synthesized from themesotetra-2-aminophenyl porphyrin which was obtained through PorphyrinProducts, Inc. Logan, Utah) and PcSi vinyl (example 4) were incorporatedinto 67 nm latex particles (polystyrene sulfate latex from InterfacialDynamics Inc., Portland, Oreg.) using the tetrahydrofuran method 1 ofexample 10. The molar ratio of the Tdap to the PcSi vinyl varied from1/1 to 2/1 to 6/1 in the latex loading solutions while maintaining aconstant mass (0.1 mg/ml) of PcSi vinyl in each solution. The dialyzedparticles were diluted to 0.0019% solids in water and the fluorescenceintensity at 680 urn of the PcSi vinyl was measured as a function ofexcitation wavelength between 350 nm and 470 nm. The excitation maximumof the Tdap is 430 nm and of the PcSi vinyl is 350 nm. The emissionmaximum of the Tdap is 650 nm. The table below shows the results.Excitation Fluorescence Tdap/PcSi vinyl λ (nm) Intensity at 680 nm 1/1350 490 1/1 430 83 1/1 450 38 1/1 470 11 2/1 350 580 2/1 430 830 2/1 450460 2/1 470 220 6/1 350 600 6/1 430 1800 6/1 450 800 6/1 470 200

These results show that as the molar ratio of donor to acceptor in thelatex particles increases from 1/1 to 6/1, the energy transfer, asmeasured by the fluorescence intensity of the acceptor dye, becomessignificantly more efficient. There was no observable emission of theTdap dye in the particles at the emission maximum of 650 nm suggestingthat the energy transfer is very efficient. The data indicates that thefluorescence intensity of the latex particles, generated through anenergy transfer pathway, is affected by the “light gathering” capabilityof the donor dye. Thus, optimization of the fluorescence intensity ofthe latex particles should involve changing the molar ratio of donor toacceptor.

EXAMPLE 14 Effect of Incorporation of Different Dyes on Quenching andFluorescence Intensity of Latex Particles

Five different silicon phthalocyanines, synthesized as described inexamples 2-6, were incorporated into 67 nm surfactant-free, polystyrenelatex particles (Interfacial Dynamics Corp. Inc. Portland, Oreg.) insets of 1, 3 or 5 dyes according to the following methods. Each siliconphthalocyanine derivative had maximum excitation and emissionwavelengths at 350 nm and 680 nm, respectively. After preparation ofeach dye-latex, each suspension was diluted to 0.057% solids in eitherwater or tetrahydrofuran. The dye-latex solutions were excited at 350 nmand the fluorescence intensity at 680 nm was measured. The intensity offluorescence in water divided by the intensity of fluorescence intetrahydrofuran minus 1 is the degree of quenching of the dyes in thelatex particles.

Preparation of One Phthalocyanine Dye in Latex

A solution of PcSi pentafluoro dye (0.02 mg) in tetrahydrofuran (0.1 ml)was added dropwise over 5 minutes to a stirred 2% solids solution oflatex particles (1.0 ml). The latex suspension was stirred at roomtemperature for 6 hours, then transferred to dialysis tubing(Spectra-por, 12-14,000 molecular weight cutoff, Spectrum, Houston,Tex.) and the dye-latex solution was dialyzed against water for 12-15hours at 4° C. The dye-latex solution was removed from dialysis and thesolids concentration was adjusted to 1.6%.

Preparation of Three Phthalocyanine Dyes in Latex

A solution which consists of PcSi pentafluoro, PcSi trihexyl and PcSicyano dyes in equimolar amounts to total 0.02 mg dye in tetrahydrofuran(0.1 ml), was added dropwise over 5 minutes to a stirred 2% solidssolution of latex particles (1.0 ml). The latex suspension was stirredat room temperature for 6 hours, then transferred to dialysis tubing(Spectra-por, 12-4,000 molecular weight cutoff, Spectrum, Houston, Tex.)and the dye-latex solution was dialyzed against water for 12-15 hours at4° C. The dye-latex solution was removed from dialysis and the solidsconcentration was adjusted to 1.6%.

Preparation of Five Phthalocyanine Dyes in Latex

A solution which consists of PcSi pentafluoro, PcSi trihexyl, PcSicyano, PcSi vinyl and PcSi methyl ester dyes in equimolar amounts tototal 0.02 mg dye in tetrahydrofuran (0.1 ml), was added dropwise over 5minutes to a stirred 2% solids solution of latex particles solution (1.0ml). The latex suspension was stirred at room temperature for 6 hours,then transferred to dialysis tubing (Spectra-por, 12-14,000 molecularweight cutoff, Spectrum, Houston, Tex.) and the dye-latex solution wasdialyzed against water for 12-15 hours at 4° C. The dye-latex solutionswere removed from dialysis and the % solids concentration was adjustedto 1.6%.

The table that follows illustrates the results of the fluorescenceexperiments. Dyes Entrapped Intensity % Quenching 1 413 72 3 561 56 5747 49

The data show that as the number of different dyes entrapped into thelatex goes from 1 to 3 to 5, the fluorescence intensity increasesbecause the quenching in the particles decreases.

EXAMPLE 15 Preparation and Characterization of Fluorescence EnergyTransfer Dye Latex (Table 1) and Fluorescent Latex Incorporating HybridPhthalocyanine Derivatives (Table 2)

A variety of fluorescent energy transfer latexes were prepared withvarious donor and acceptor dye molecules. Table 1 shows the loadingconcentrations of the respective donor and acceptor dyes, the mole ratioof the donor and acceptor dyes, the dye loading solvent system asdescribed in Example 10 and the excitation and emission wavelengths andthe fluorescence intensity for each particle size at the specifiedsolids concentration. For some of the energy transfer latexes, the samedye pair was incorporated into different diameter latexes. Thefluorescence energy transfer efficiency of the entries is greater than80%. The dye system represented in line 56 is a fluorescence energytransfer compound (FET compound) so that the donor and acceptor pairreside in the molecule before incorporation into latex.

Table 2 shows the characteristics of latex particles incorporated withhybrid phthalocyanine derivatives as described in Example 10 and thefluorescence intensity at the specified solids concentration. TABLE 1Emission Loading Conc. Loading Mole Donor:Mole Solvent System Intensity(% Maximum Donor Dye (mg/ml) Acceptor Dye Conc. (mg/ml) Acceptor (LatexSize) solid) (Excit.) 1. trans-4-[4-(Dibutyl amino) styryl]- 0.12 mg/mlSilicon pthalocyanine 0.1 mg/ml 2:1 THF (20%) 340 679 nm (475 nm)1-methyl pyridinium iodide bis(dimethyl-vinylsilyloxide) (0.067 μm)(0.0019%) 2. trans-4-[4-(Dibutyl amino) styryl]- 0.1 mg/ml Silicon2,3-naphthalocyanine 0.23 mg/ml 1:1 DMF (70%) 347 789 nm (475 nm)1-methyl pyridinium iodide bis(dimethyl-vinylsilyloxide) (0.067 μm)90.057%) 3. trans-4-[4-(Dibutyl amino) styryl]- 0.1 mg/ml1,1′-Dihexl-3,3,3′,3′- 0.144 mg/ml 1:1 DMF (70%) 688 688 nm (645 nm)1-methyl pyridinium iodide tetramethylindodicarbo-cynanine (0.067 μm)(0.057%) oxide 4. Meso-tetra-2-aminophenyl 0.18 mg/ml Siliconphthalocyanine 0.1 mg/ml 2:1 THF (20%) 1000 679 nm (420 nm) porphinebis(dimethylvinylsilyloxide) (0.202 μm) (0.00095%) 5.Meso-tetra-2-aminophenyl 0.1 mg/ml 1,1′-Dihexyl-3,3,3′,3′- 0.098 mg/ml1:1 DMF (70%) 157 676 nm (645 nm) porphine tetramethylindodicarbocyanine(0.067 μm) (0.0019%) iodide 6. Meso-tetra-2- 0.21 mg/ml Siliconphtalocyanine 0.1 mg/ml 2:1 THF 920%) 209 679 nm (430 nm)dimethylaminophenyl porphine bis(dimethylvinylsilyloxide) (0.412 μm)(0.00095%) 7. 3-Ethyl-3′ethyl 0.056 mg/ml Silicon 2,3-naphthalocynanine0.35 mg/ml 4:1 DMF (70% 289 (0.057%) 785 nm (650 mn)carboxyethylthiadicarbocynanine bis(dimethylvinylsilyloxide) (0.067 μm)iodide 8. 1,1′-Dioctadecly-3,3,3,3′,3′- 0.036 mg/ml Silicon2,3-naphthalocynanine 0.013 mg/ml 4:1 DMF (70%) 324 787 nm (650 nm)tetramethylindodicarbocyanine bis(dimethylvinylsilyloxide) (0.0067 μm)(0.057%) perchlorate 9. 1,1′-Diethyl-3,3,3′,3′- 0.078 mg/ml Silicon2,3-naphthalocyanine 0.025 mg/ml 6:1 DMF (70%) 723 787 mn (635 nm)tetramethylindodicarbocyanine bis(dimethylvinylsilyloxide) (0.067 μm)(0.057%) iodide 10. 1,1′-Dihexyl-3,3,3′3′- 0.095 mg/ml Silicon2,3-naphthalocynanine 0.025 mg/ml 6:1 DMF (70%) 907 783 nm (635 nm)tetramethylindodicarbocynanine bis(dimethylvinylsilyloxide) (0.067 μm)(0.057%) iodide 11. 3,3′-Diethyl thiatricarbocynanine 0.013 mg/mlSilicon 2,3-naphthalocynanine 0.025 mg/ml 1:1 DMF (70%) 12 788 nm (650nm) iodide bis(dimethylvinylsilyloxide) (0.067 μm) (0.057%) 12.3,3″-Dipropyl 0.013 mg/ml Silicon 2,3-naphthalocyanine 0.025 mg/ml 1:1DMF (70%) 65 788 nm (660 nm) thiadicarbocyanine iodidebis(dimethylvinylsilyloxide) (0.067 μm) (0.057%) 13.1,9-Dimethyl-methylene blue, 0.008 mg/ml Silicon 2,3-naphthalocyanine0.025 mg/ml 1:1 DMF (70%) 57 788 nm (650 nm) chloridebis(dimethylvinylsilyloxide) (0.067 μm) (0.057%) 14.N,N′-Di(3-trimethyl- 0.013 mg/ml Silicon 2,3-naphthalocyanine 0.025mg/ml 1:1 DMF (70%) 63 788 nm (650 nm) ammoniumpropyl)thiadicarbocyanine bis(dimethylvinylsilyloxide) (0.067 μm) (0.057%)tribomide 15. 1,1′,3,3,3′,3′- 0.012 mg/ml Silicon 2,3-naphthalocyaninebis 0.025 mg/ml 1:1 DMF (70%) 33 788 nm (650 nm)Hexamethylindotricarbocyanine (dimethylvinylsilyloxide) (0.067 μm)(0.057%) perchlorate 16. N-(3-Triethyl-ammoniumpropyl)- 0.014 mg/mlSilicon 2,3-naphthalocyanine bis 0.025 mg/ml 1:1 DMF (70%) 55 (0.0575)788 nm (500 nm) 4-(4-(pdibutylaminophenyl) (dimethylvinylsilyloxide)(0.067 μm) butadienyl) pyridium, dibromide 17. 1,1′3,3,3′,3′-Hexamethyl-0.015 mg/ml Silicon 2,3-naphthalocyanine bis 0.025 mg/ml 1:1 DMF (70%) 8(0.0575) 788 nm (650 nm) 4,4′,5,5′-dibenzo-2,2′-indotricarbocyanine(dimethylvinylsilyloxide) (0.067 μm) perchlorate 18. Fluoroscein 0.264mg/ml Silicon phthalocyanine 0.1 mg/ml 6:1 THF (20% (0.067 μm) 517 683nm (485 nm) bis(dimethylvinylsilyloxide) (0.057%) 19. Chlorophyll B0.087 mg/ml Silicon 2,3-naphthalocyanine 0.025 mg/ml 4:1 THF (20%) 72785 nm (440 nm) bis(dimethylvinylsilyloxide) (0.067 μm) (0.057%) 20.Chlorophyll B 0.244 mg/ml Silicon phthalocyanine 0.1 mg/ml 2:1 THF (20%)140 679 nm (440 nm) bis(dimethylvinylsilyloxide) (0.067 μm) (0.0019%)21. trans-4-[4-(Dibutyl amino) 0.181 mg/ml Silicon phthalocyanine 0.07mg/ml 4:1:1 THF (20%) 300 681 nm (475 nm) styryl]-1-methyl pyridiniumiodide bis(dimethylpentafluorophenylsilyloxide) + Silicon 0.05 mg/ml(0.067 μm) (0.0019%) phthalocyanine bis(dimethylvinylsilyloxide) 22.trans-4-[4-Dibutyl amino) styryl]- 0.072 mg/ml Silicon phthalocyanine0.04 mg/ml 4:1:1:1 THF (20%) 206 681 nm (475 nm) 1-methyl pyridinumiodide bis(trihexylsilyloxide) + Silicon 0.04 mg/ml (0.067 μm) (0.0019%)phthalocyanine 0.03 mg/ml bis(dimethylpentafluorophenylsilyloxide) +Silicon pthalocyanine bis(dimethylvinylsilyloxide) 23.3-Ethyl-3′-carboxyethylthiadicarbocyanine 0.013 mg/ml Silicon2,3-naphthalocyanine bis 0.025 mg/ml 1:1 DMF (70%) 76 788 nm (625 nm)iodide (dimethylvinylsilyloxide) (0.067 μm) (0.057%) 24.3-Ethyl-3′-ethyl- 0.013 mg/ml Silicon 2,3-naphthalocyanine bis 0.025mg/ml 1:1 DMF (70%) 135 788 nm (630 nm) carboxyethloxathiadicarbocyanine(dimethylvinylsilyloxide) (0.067 μm) (0.057%) iodide 25.3,3′-Diethylthia-dicarbocyanine 0.013 mg/ml Silicon 2,3-naphthalocyanine0.025 mg/ml 1:1 DMF (70%) 59 787 nm (660 nm) iodide bis(dimethylvinylsilyloxide) (0.067 μnm) (0.057%) 26.3,3′-Diethyloxa-dicarbocyanine 0.012 mg/ml Silicon 2,3-naphthalocyanine0.025 mg/ml 1:1 DMF (70%) 57 787 nm (590 nm) iodide bis(dimethylvinylsilyloxide) (0.067 μm) (0.057%) 27.1,1′-Dihexyl-3,3,3′,3′- 0.094 mg/ml Silicon 2,3-naohthalocyanine bis0.026 mg/ml 6:1:2 DMF (50% 127 788 nm (650 nm)tetramethylindodicarbocyanine (dimethylvinylsilyloxide) + Silicon 0.05mg/ml (0.431 μm CMLI) (0.057%) iodide naphthlocyanine bis(dimethylethyl- maleimidosilyloxide 28. 1,1′-Dihexyl-3,3,3′,3′- 0.094mg/ml Silicon 2,3-naphthalocyanine 0.025 mg/ml 6:1:2 DMF (50% 193 788 nm(635 nm) tetramethylindodicarbocyanine bis(dimethylvinylsilyloxide) +Silicon 0.05 mg/ml (0.431 μm CML) (0.057%) iodide phthalocyanine bis(dimethylethyl- maleimidosilyloxide) 29. 1,1′-Dihexyl-3,3,3′,3′- 0.03mg/ml Silicon 2,3-naphthalocyanine 0.05 mg/ml 1:1 DMF (50%) 275 788 nm(650 nm) tetramethylindodicarbocyanine bis(dimethylvinylsilyloxide)(0.431 μm CML) (0.057%) iodide 30. 1,1′-Dihexyl-3,3,3′,3′- 0.1 mg/mlSilicon 2,3 naphthalocyanine bis 0.2 mg/ml 1:1 DMF (50%) 163 798 nm (650nm) tetramethylindodicarbocyanine (dimethyltriphenylsilyloxide) (0.431μm CML) (0.057%) iodide 31. 1,1′-Dihexyl-3,3,3′,3′- 0.09 mg/ml Siliconnaphthalocyanine bis 0.05 mg/ml 4:1 DMF (50%) 153 790 nm (650 nm)tetramethylindodicarbocyanine (dimethylretinol) (0.431 μm CML) (0.057%)iodide 32. 1,1′,3,3,3′,3′- 0.216 mg/ml Silicon 2,3 naphthalocyanine bis0.1 mg/ml 4:1 DMF (50% 0.431 μm 0.4 788 nm (635 nm)Hexamethylindodicarbocyanine (dimethyltriphenylsilyloxide) CML)(0.00057%) perchlorate 33. 1,1′-Dihexyl-3,3,3′,3′- 0.512 mg/ml1,1′,3,3,3′,3′- 0.1 mg/ml 4:1 DMF (50%) 0.9 776 nm (635 nm)tetramethylindodicarbocyanine Hexamethylindotridicarbocyanine (0.431 μmCML) (0.00057%) iodide perchlorate 34. Lithium tetracetylide boron 0.16mg/ml Silicon 2,3 naphthalocyanine bis 0.1 mg/ml 4:1 DMF (50% 22 788 nm9635 nm) complex of 1,1′-Dihexyl-3-3,3′,3′-(dimethylhexlvinylsilyloxide) (0.216 μm CML) (0.00057%)tetramethylindo-dicarbocyanine iodide 35. Silicon phthalocyanine 0.334mg/ml Silicon 2,3 naphthalocyanine bis 0.1 mg/ml 10:1  DMF (50% 1 800 nm(650 nm) bis(dimethylvinylsilyloxide) (dimethylhexlvinylsilyloxide)(0.216 μm CML) (0.00057%0 36. 1,1′,3,3,3′,3′- 0.23 mg/ml Silicon 2,3naphthalocyanine bis 0.1 mg/ml 10:1  DMF (50%) 0.4 780 nm (635 nm)Hexamethylindotricarbocyanine (dimethylhexlvinylsilyloxide) (0.216 μmCML) (0.0005%) perchlorate 37. 1,1′,3,3,3′,3′- 0.19 mg/ml Siliconoctaethoxy 2,3- 0.1 mg/ml 10:1  DMF (50%) 0.7 780 nm (635 nm)Hexamethylindotridicarbocyanine naphthalocyanine bis(dimethylhexylvinylsilyloxide) (0.216 μm CML) (0.00057%0 perchlorate 38.Oxazine 1 perchlorate 0.01 mg/ml Silicon 2,3-naphthalocyanine bis 0.025mg/ml 1:1 DMF (70%) 291 788 nm (650 nm) (dimethylhexlvinylsilyloxide)(0.067 μm) (0.057%) 39. 3,3′-Dipropyl- 0.232 mg/ml Silicon2,3-naphthalocyanine bis 0.1 mg/ml 4:1 DMF (50%) 0.4 788 nm (635 nm)thiadicarbocyanine iodide (dimethylvinylsilyloxide) (0.431 μm CML)(0.00057%) 40. Copper tetra-tert-butylphthalocyanine 0.72 mg/ml Silicon2,3-naphthalocyanine bis 0.1 mg/ml 1:1 DMF (50%) 0.2 788 nm (650 nm)(dimethyl-hexylvinylsilyloxide) (0.216 μm CML) (0.00057%) 41.(E,E)-3,5-bis-(4-phenyl-1,3- 0.16 mg/ml Silicon 2,3-naphthalocyanine bis0.1 mg/ml 4:1 DMF (50%) 42 785 nm (670 nm)butadienyl)-4,4-difluoro-4-bora- (dimethylhexylvinylsilyloxide) (0.216μm CML) (0.00057%) 3a,4a-diazo-s-indacene 42. Aluminum tetra-tert-butyl0.28 mg/ml Silicon 2,3-naphthalocyanine bis 0.1 mg/ml 4:1 THF (50%) 05788 nm (650 nm) phthalocyanine hydroxide (dimethylhexylvinylsilyloxide)(0.216 μm CML) (0.00057%) 43. Aluminum tetra-tert- 0.29 mg/ml Silicon2,3-naphthalocyanine bis 0.1 mg/ml 4:1 DMF (50%) 0.1 788 nm (650 nm)butylphthalocyanine chloride (dimethylhexylvinylsilyloxide) (0.216 μmCML) (0.00057%) 44. (E,E)-3,5-bis-(4-phenyl-1,3- 0.14 mg/ml Aluminumoctabutoxy- 0.1 mg/ml 4:1 THF (50%) 1.8 774 nm (650 nm)butadienyl)-4,4-difluoro-4-bora- phthalocyanine triethyisilyloxide(0.216 μm CML) (0.00057%) 3a,4a-diazo-s-indacene 45. Iron phthalocyaninebis(tert- 0.26 mg/ml Silicon 2,3-naphthalocyanine bis 0.1 mg/ml 4:1 THF(50%) 0.3 788 nm (670 nm) butyl isocyanide)(dimethylhexylvinylsilyloxide) (0.216 μm CML) (0.00057%) 46.(E,E)-3,5-bis-(4-phenyl-1,3- 0.16 mg/ml Octabutoxy-phthalocyanine 0.1mg/ml 4:1 THF (50%) 0.7 783 nm (670 nm) butadienyl)-4,4-difluoro-4-bora-(0.216 μm CML) (0.00057%) 3a,4a-diazo-s-indacene 47.(E,E)-3,5-bis-(4-phenyl-1,3- 0.15 mg/ml Silicon 2,3-naphthalocyanine bis0.1 mg/ml 4:1 THF (50%) 16.9 783 nm (670 nm)butadienyl)-4,4-difluoro-4-bora- (dimethylphenylpentafluoro- (0.216) μmCML) (0.00057%) 3a,4a-diazo-s-indacene silyloxide) 48.(E,E)-3,5-bis-(4-phenyl-1,3- 0.19 mg/ml Silicon 2,3-naphthalocyanine bis0.1 mg/ml 4:1 THF (50%) 31.5 783 nm (670 nm)butadienyl)-4,4-difluoro-4-bora- (dimethylvinylisilyloxide) (0.216 μmCML) (0.00057%) 3a,4a-diazo-s-indacene 49. (E,E)-3,5-bis-(4-phenyl-1,3-0.15 mg/ml Silicon 2,3-naphthalocyanine bis 0.1 mg/ml 4:1 THF (50%) 13.1783 nm (670 nm) butadienyl)-4,4-difluoro-4-bora-(diphenylvinylsilyloxide) (0.216 μm CML) (0.00057%)3a,4a-diazo-s-indacene 50. (E,E)-3,5-bis-(4-phenyl-1,3- 0.15 mg/mlSilicon 2,3-naphthalocyanine bis 0.1 mg/ml 4:1 THF (50%) 4.7 783 nm (670nm) butadienyl)-4,4-difluoro-4-bora- (dimethylmalemidoethoxysilyloxide)(0.216 μm CML) (0.00057%) 3a,4a-diazo-s-indacene 51.(E,E)-3,5-bis-(4-phenyl-1,3- 0.14 mg/ml Silicon 2,3-naphthalocyanine bis0.1 mg/ml 4:1 THF (50%) 11.7 783 nm (670 nm)butadienyl)-4,4-difluoro-4-bora- (dimethylsilyloxide-trans- (0.216 μmCML) (0.00057%) 3a,4a-diazo-s-indacene stilbene)) 52.(E,E)-3,5-bis-(4-phenyl-1,3- 0.12 mg/ml Silicon 2,3-naphthalocyanine 0.1mg/ml 4:1 THF (50%) 22.3 783 nm (670 nm)butadienyl)-4,4-difluoro-4-bora- bis(tri-decafluoro-1,1,2,2-tetra-(0.216 μm CML) (0.00057%) 3a,4a-diazo-s-indacenehydrococtyl-1-dimethyl-silyloxide) 53. (E,E)-3,5-bis-(4-phenyl-1,3- 0.12mg/ml Silicon 2,3-naphthalocyanine 0.1 mg/ml 4:1 THF (50%) 16.1 783 nm(670 nm) butadienyl)-4,4-difluoro-4-bora- bis(dimethylretinol) (0.216 μmCML) (0.00057%) 3a,4a-diazo-s-indacene 54. Germanium tetra-tert-butyl0.3 mg/ml Silicon 2,3-naphthalocyanine bis 0.1 mg/ml 4:1 THF (50%) 1.3783 nm (670 nm) phthalocyanine dihydroxide(dimethylhexylvinylsilyloxide) (0.216 μm CML) (0.00057%) 55. Germanumtetra-tert-butyl 0.3 mg/ml Silicon 2,3-naphthalocyanine bis 0.1 mg/ml4:1 THF (50%) 0.6 783 nm (670 nm)5 phthalocyanine dichloride(dimethylhexylvinylsilyloxide) (0.216 μm CML) (0.00057%) 56. Siliconphthalocyanine bis 0.15 mg/ml Silicon phthalocyanine THF (20%) 209 681nm (470 nm) (maleimide-fluoroscein) FET bis(maleimide-fluoroscein) FET(0.067 μm) (0.0019%) COMPOUND COMPOUND 57.3,3′-Diethylthia-tricarbocyanine 0.57 mg/ml5,5′-Dichloro-1′-diphenylamino- 0.1 mg/ml 4:1 DMF (50%) 0.048 nA 832 nm(670 nm) 3,3′-diethyl-10,12- (0.216 μm CML) (0.00057%)ethylenethiatricarbocyanine iodide 58. 1,1′,3,3,3′,3′- 0.61 mg/ml5,5′-Dichloro-1,1′-diphenylamino- 0.1 mg/ml 4:1 DMF (50%) 0.149 nA 832nm (670 nm) Hexamethylindoincarbocyanine 3,3′-diethyl-10,12- (0.216 μmCML) (0.00057%) ethylenethiatricarbocyanine iodide 59.1,1′,3,3,3′,3′-Hexamethyl- 0.51 mg/ml 5,5′-Dichloro-1,1′-diphenylamino-0.1 mg/ml 4:1 DMF (50%) 0.046 nA 832 nm (670 nm)4,4′,5,5′-dibenzo-2,2′-indotricarbocyanine 3,3′-diethyl-10,12- (0.216 μmCML) (0.00057%) perchlorate ethylenethiatricarbocyanine iodide 60.1,1′-Dihexyl-3,3,3′,3′- 0.23 mg/ml Silicon 2,3-naphthalocyanine bis 0.1mg/ml 4:1 DMF (50%) 14.12 nA 783 nm (670 nm)tetramethylindodicarbocyanine (dimethylhexylvinylsilyloxide) (0.216 μmCML) (0.00057%) iodide 61. (E,E)-3,5-bis-(4-phenyl-1,3- 0.16 mg/mlSilicon 2,3-naphthalocyanine bis 0.1 mg/ml 4:1 DMF (50%) 5.00 nA 783 nm(670 nm) butadienyl)-4,4-difluoro-4-bora- (dimethylhexylvinylsilyloxide)(0.216 μm CML) (0.00057%) 3a,4a-diazo-s-indacene 62.(E,E)-3,5-bis-(4-phenyl-1,3- 0.26 mg/ml Silicon octaethoxy 2,3- 0.1mg/ml 4:1 DMF (50%) 2.74 nA 858 nm (670 nm)butadienyl)-4,4-difluoro-4-bora- naphthalocyanine bis(dimethylhexylvinylsilyloxide) (0.216 μm CML) (0.00057%)3a,4a-diazo-s-indacene 63. (E,E)-3,5-bis-(4-phenyl-1,3- 0.32 mg/mlOctabutoxy-phthalocyanine 0.1 mg/ml 4:1 DMF (50%) 4.07 nA 762 nm (670nm) butadienyl)-4,4-difluoro-4-bora- (0.216 μm CML) (0.00057%)3a,4a-diazo-s-indacene 64. (E,E)-3,5-bis-(4-phenyl-1,3- 0.28 mg/mlOctabutoxy-naphthalocyanine 0.1 mg/ml 4:1 DMF (50%) 1.76 nA 772 nm (670nm) butadienyl)-4,4-difluoro-4-bora- (0.216 μm CML) (0.00057%)3a,4a-diazo-s-indacene 65. 1,1′-Dihexyl-3,3,3′,3′- 0.19 mg/ml Siliconoctaethoxy 2,3- 0.1 mg/ml 4:1 DMF (50%) 0.712 nA 858 nm (670 nm)tetramethylindodicarbocyanine naphthalocyanine bis (0.216 μm CML)(0.00057%) iodide (di-methylhexylvinylsilyloxide) 66.3,3′-Diethylthia-tricarbocyanine 0.16 mg/ml Silicon octaethoxy 2,3- 0.1mg/ml 4:1 DMF (50%) 0.58 nA 858 nm (670 nm) naphthalocyanine bis (0.216μm CML) (0.00057%) (di-methylhexylvinylsilyloxide) 67. 1,1′,3,3,3′,3′-0.15 mg/ml Silicon octaethoxy 2,3- 0.1 mg/ml 4:1 DMF (50%) 0.141 nA 858nm (670 nm) Hexamethylindotricarbocyanine naphthalocyanine bis (0.216 μmCML) (0.00057%) (dimethylhexylvinylsilyloxide) 68.1,1′,3,3,3′,3′-Hexamethyl- 0.19 mg/ml Silicon octaethoxy 2,3- 0.1 mg/ml4:1 DMF (50%) 0.058 nA 858 nm (670 nm)4,4′,5,5′-dibenzo-2,2′-Indotricarbocyanine naphthalocyanine bis (0.216μm CML) (0.00057%) perchlorate (di-methylhexylvinylsilyloxide) 69.(E,E)-3,5-bis-(4-phenyl-1,3- 0.2 mg/ml Silicon octaethoxy 2,3- 0.15mg/ml 4:1 THF (50%) 2.720 nA 858 nm (670 nm)butadienyl)-4,4-difluoro-4-bora- naphthalocyanine bis (0.216 μm CML)(0.00057%) 3a,4a-diazo-s-indacene (di-methylhexylvinylsilyloxide) 70.(E,E)-3,5-bis-(4-phenyl-1,3- 0.16 mg/ml Silicon octaethoxy 2,3- 0.1mg/ml 4:1:1 THF (50%) 2.38 nA 858 nm (670 nm)butadienyl)-4,4-difluoro-4-bora- naphthalocyanine bis 0.12 mg/ml (0.216μm CML) (0.00057%) 3a,4a-diazo-s-indacene(dimethylhexylvinylsilyloxide) + Silicon octaethoxy 2,3-naphthalocyanine bis (dimethylhexylvinylsilyloxide) 71. Siliconphthalocyanine 0.36 mg/ml 5,5′-Dichloro-1,1′- 0.1 mg/ml 4:1 THF (50%)8.10 nA 832 nm (670 nm) bis(di-methylvinlsilyloxide)diphenylamino-3,3′-diethyl- (0.216 μm CML) (0.00057%) 10,12-ethylenethiatricarbocyanine perchlorate 72. Tetrakis(4-cumyl-phenoxy)0.48 mg/ml Silicon 2,3-naphthalocyanine bis 0.1 mg/ml 4:1 THF (50%)0.397 nA 783 nm (670 nm) phthalocyanine (dimethylhexylvinylsilyloxide)(0.216 μm CML) (0.00057%) 73. Tetrakis(4-cumyl-phenoxy) 0.68 mg/ml5,5′-Dichloro-1,1′- 0.1 mg/ml 4:1 THF (50%) 0.128 nA 832 nm (670 nm)phthalocyanine diphenylamino-3,3′-diethyl- (0.216 μm CML) (0.00057%)10,12- ethylenethiatricarbocyanine perchlorate 74. Tetrakis(phenylthio)0.34 mg/ml Silicon 2,3-naphthalocyanine bis 0.1 mg/ml 4:1 THF (50%)0.374 nA 788 nm (670 nm) phthalocyanine (dimethylhexylvinylsilyloxide)(0.216 μm CML) (0.00057%) 75. Tetrakis(phenylthio) 0.28 mg/ml5,5′-Dichloro-1,1′- 0.1 mg/ml 4:1 THF (50%) 0.109 nA 832 nm (670 nm)phthalocyanine diphenylamino-3,3′-diethyl- (0.216 μm CML) (0.00057%)10,12-ethylenethiatricarbocyanine perchlorate 76.(E,E)-3,5-bis-(4-phenyl-1,3- 0.24 mg/ml Tin octabutoxy 2,3- 0.1 mg/ml4:1 THF (50%) 1.724 nA >900 nm (670 nm) butadienyl)-4,4-difluoro-4-bora-naphthalocyanine dichloride (0.216 μm CML) (0.00057%)3a,4a-diazo-s-indacene 77. Tetrakis (4-cumylphenoxy) 0.36 mg/ml Tinoctabutoxy 2,3- 0.1 mg/ml 4:1 THF (50%) 0.162 nA >900 nm (670 nm)phthalocyanine naphthalocyanine dichloride (0.216 μm CML) (0.00057%) 78.Tetrakis(phenylthio) 0.26 mg/ml Tin octabutoxy 2,3- 0.1 mg/ml 4:1 THF(50%) 0.061 nA >900 nm (670 nm) phthalocyanine naphthalocyaninedichloride (0.216 μm CML) (0.00057%) 79. Germanium tetra-tert-butyl 0.42mg/ml 5,5′-Dichloro-1,1′- 0.1 mg/ml 4:1 THF (50%) 0.109 nA >900 nm (670nm) phthalocyanine dihydroxide diphenylamino-3,3′-diethyl- (0.216 μmCML) (9.00057%) 10,12- ethylenethiatricarbocyanine perchlorate 80.Germanium tetra-tert-butyl 0.22 mg/ml Tin octabutoxy 2,3- 0.1 mg/ml 4:1THF (50%) 0.045 nA >900 nm (670 nm) phthalocyanine dihydroxidenaphthalocyanine dichloride (0.216 μm CML) (0.00057%) 81. Germaniumtetra-tert-butyl 0.2 mg/ml Tin octabutoxy 2,3- 0.1 mg/ml 4:1 THF (50%)0.042 nA >900 nm (670 nm) phthalocyanine dihydroxide naphthalocyaninebis (0.216 μm CML) (0.00057%) (triethylsilyloxide) 82. Germaniumtetra-tert-butyl 0.42 mg/ml 5,5′-Dichloro-1,1′- 0.1 mg/ml 4:1 THF (50%)0.081 nA 832 nm (670 nm) phthalocyanine dichloridediphenylamino-3,3′-diethyl- (0.216 μm CML) (0.00057%)10,12-ethylenethiatricarbocyanine perchlorate 83. Germaniumtetra-tert-butyl 0.22 mg/ml Tin octabutoxy 2,3- 0.1 mg/ml 4:1 THF (50%)0.052 nA >900 nm (670 nm) phthalocyanine dichloride naphthalocyaninedichloride (0.216 μm CML) (0.00057%) 84. Germanium tetra-tert-butyl 0.2mg/ml Tin octabutoxy 2,3- 0.1 mg/ml 4:1 THF (50%) 0.050 nA >900 nm (670nm) phthalocyanine dichloride naphthalocyanine bis (0.216 μm CML)(0.00057%) (triethylsilyloxide) 85. (E,E)-3,5-bis-(4-phenyl-1,3- 0.16mg/ml Silicon 2,3-naphthalocyanine bis 0.1 mg/ml 4:1:1 THF (50%) 0.315nA 858 nm (670 nm) butadienyl)-4,4-difluoro-4-bora-(dimethylhexylvinylsilyloxide) + 5,5′- 0.072 mg/ml (0.216 μm CML)(0.00057%) 3a,4a-diazo-s-indacene Dichloro-1,1′-diphenylamino-3,3′-diethyl- 10,12-ethylenethiatricarbocyanineperchlorate 86. (E,E)-3,5-bis-(4-phenyl-1,3- 0.24 mg/ml5,5′-Dichloro-1,1′- 0.1 mg/ml 4:1 THF (50%) 2.230 nA 832 nm (670 nm)butadienyl)-4,4-difluoro-4-bora- diphenylamino-3,3′-diethyl- (0.216 μmCML) (0.00057%) 3a,4a-diazo-s-indacene 10,12-ethylenethiatricarbocyanineperchlorate 87. 1,1′-Dihexyl-3,3,3′,3′- 0.34 mg/ml 5,5′-Dichloro-1,1′-0.1 mg/ml 4:1 THF (50%) 0.545 nA 823 nm (670 nm)tetramethylindodicarbocyanine diphenylamino-3,3′-diethyl- (0.216 μm CML)(0.00057%) iodide 10,12-ethylenethiatricarbocyanine perchlorate 88.(E,E)-3,5-bis-(4-phenyl-1,3- 0.16 mg/ml Silicon octaethoxy 2,3- 0.07mg/ml 4:1:1 THF (50%) 49 783 nm (670 nm)butadienyl)-4,4-difluoro-4-bora- naphthalocyanine bis 0.07 mg/ml (0.216μm CML) (0.00057%) 3a,4a-diazo-s-indacene(dimethylhexylvinylsilyloxide) + Silicon octaethoxy 2,3-naphthalocyanine bis (dimethylpentafluoro phenylsilyloxide) 89. Siliconphthalocyanine 1.0 mg/ml Silicon octaethoxy 2,3- 1.0 mg/ml 1:5:1 THF(50%) 0.4 858 nm (670 nm) bis(dimethylhexylvinylsilyloxide)naphthalocyanine bis (dimethylhexylvinylsilyloxide) (0.216 μm CML)(0.00057%) 90. Silicon phthalocyanine 1.0 mg/ml Silicon2,3-naphthalocyanine bis 1.0 mg/ml 1:5:1.2:1 THF (50%) 0.4 854 nm (670nm) bis(dimethylhexylvinylsilyloxide) (dimethylhexylvinylsilyloxide) +Silicon 1.0 mg/ml (0.216 μm CML) (0.00057%) octaethoxy 2,3-naphthalocyanine bis (dimethylhexylvinylsilyloxide) 91. Siliconphthalocyanine bis 1.0 mg/ml Silicon 2,3-naphthalocyanine bis 0.1 mg/ml9:7:1 THF (50%) 155.8 785 nm (670 nm) (Inhexylsilyloxide)(dimethylhexylvinylsilyloxide) (0.216 μm CML) (0.00057%) 92. Siliconphthalocyanine bis(3- 1.0 mg/ml Silicon 2,3-naphthalocyanine bis 0.1mg/ml 13:5:1  THF (50%) 23.2 785 nmm cyanopropyl) (dimethylsilyloxide)(dimethylhexylvinylsilyloxide) (0.216 μm CML) (0.00057%) (670 nm) 93.Silicon phthalocyanine bis 1.0 mg/ml Silicon 2,3-naphthalocyanine bis0.1 mg/ml 10:1:1  THF (50%) 14.5 785 nm (6670 nm)(dimethylpentafluorophenylsilyloxide) (dimethylhexylvinylsilyloxide)(0.216 μm CML) (0.00057%) 94. Silicon phthalocyanine 1.0 mg/ml Silicon2,3-naphthalocyanine bis 0.1 mg/ml 10:3:1  THF (50%) 70.5 785 nm (670nm) dimethylpentafluorophenylsilyloxide (dimethylhexylvinylsilyloxide)(0.216 μm CML) (0.00057%) trihexylisilyloxide 95. Silicon phthalocyaninebis (10- 1.0 mg/ml Silicon 2,3-naphthalocyanine bis 0.1 mg/ml 10:2:1 THF (50%) 200.8 785 nm (670 nm) carbomethoxydecyl)(dimethylhexylvinylsilyloxide) (0.216 μm CML) (0.00057%)(dimethylsilyloxide) 96. Silicon phthalocyanine bis 1.0 mg/ml Silicon2,3-naphthalocyanine bis 0.1 mg/ml 14:7:1  THF (50%) 126.8 780 nm (670nm) (dimethylhexylvinylsilyloxide) (trihexylsilyloxide) (0.216 μm CML)(0.00057%) 97. Silicon phthalocyanine [(10- 1.0 mg/ml Silicon2,3-naphthalocyanine bis 0.1 mg/ml 12:1:1  THF (50%) 207.7 785 nm (670nm) carbomethoxydecyl) (dimethylhexylvinylsilyloxide) (0.216 μm CML)(0.00057%) dimethyisilyloxide) (dimethylvinylsilyloxide) 98. Siliconphthalocyanine bis 1.0 mg/ml Silicon 2,3-naphthalocyanine bis 0.1 mg/ml15:3:1  THF (50%) 262.8 780 nm (670 nm) (dimethylhexylvinylsilyloxide)(dimethyloctyidecylsilyloxide) (0.216 μm CML) (0.00057%) 99. Siliconphthalocyanine [(10- 1.0 mg/ml Silicon 2,3-naphthalocyanine bis 0.1mg/ml 14:6:1  THF (50%) 117.2 780 nm (670 nm) carbomethoxydecyl)(trihexylsilyloxide) (0.216 μm CML) (0.00057%) dimethyisilyloxide)(dimethylvinylsilyloxide) 100. Silicon phthalocyanine bis 1.0 mg/mlSilicon [di(1,6-diphenyl-2,3- 0.1 mg/ml 1:5:1 THF (50%) 177.6 770 nm(670 nm) (dimethylhexylvinylsilyloxide) naphthalocyanine) (0.216 μm CML)(0.00057%) phthalocyanine bis (dimethylhexylvinylsilyloxide) 101.Silicon phthalocyanine bis 1.0 mg/ml Silicon [di(1,6-diphenyl-2,3- 1.0mg/ml 1:6:1 THF (50%) 141.3 780 nm (670 nm)(dimethylhexylvinylsilyloxide) naphthalocyanine)] di(2,3-tert- (0.216 μmCML) (0.00057%) butylphthalocyanine) phthalocyanine bis(dimethylhexylvinylsilyloxide) 102. Silicon phthalocyanine bis 1.0 mg/mlSilicon [di(2,3- 1.0 mg/ml 1:4:1 THF (50%) 66.5 760 nm (670 nm)(dimethylhexylvinylsilyloxide) naphthalocyanine)] di(1,4- (0.216 μm CML)(0.00057%) diphenylphthalocyanine) bis (dimethylhexylvinylsilyloxide)103. Silicon phthalocyanine bis 1.0 mg/ml Silicon [di(1,6-diphenyl-2,3-1.0 mg/ml 1:5:1 THF (50%) 259.3 760 nm (670 nm)(dimethylhexylvinylsilyloxide) naphthalocyanine) (0.216 μm CML)(0.00057%) diphthalocyanine bis (trihexylsilyloxide) 104. Siliconphthalocyanine bis 1.0 mg/ml Silicon [di(1,6-diphenyl-2,3- 1.0 mg/ml1:5:1 THF (50%) 7.7 843 nm (670 nm) (dimethylhexylvinylsilyloxide)naphthalocyanine)] dis(2,3- (0.216 μm CML) (0.00057%)dicyanophthalocyanine) bis (dimethylhexyl-vinylsilyloxide) 105. Siliconphthalocyanine bis 1.0 mg/ml Silicon 2,3-naphthalocyanine bis 0.1 mg/ml15:1  THF (50%) 55.5 785 nm (670 nm) (dimethylvinylsilyloxide)(dimethylhexylvinylsilyloxide) (0.216 μm CML) (0.00057%) 106. Siliconphthalocyanine bis 10.4 mg/ml Silicon [di(1,6- 1.0 mg/ml 15:1:0:11 THF(70%) 503 785 nm (670 nm) (dimethylhexylvinylsilyloxide)diphenylnaphthalocyanine)] 0.1 mg/ml (0.216 μm CML) (0.00057%)diphthalocyanine bis (dimethylhexylvinylsilyloxide) + Silicon2,3-naphthalocyanine bis (dimethylhexylvinylsilyloxide). 107. Siliconphthalocyanine bis 10.4 mg/ml Silicon [di(1,6- 1.0 mg/ml 15:1  THF (70%)750 760 nm (670 nm) (dimethylhexylvinylsilyloxide)diphenylnaphthalocyanine)] (0.216 μm CML) (0.00057%) diphthalocyaninebis (dimethylhexylvinylsilyloxide) 108. Silicon phthalocyanine bis 1.2mg/ml Silicon 2,3-naphthalocyanine bis 0.1 mg/ml 15:1  THF (50%) 335 785nm (670 nm) (dimethylhexylvinylsilyloxide)(dimethylhexylvinylsilyloxide) (0.216 μm CML) (0.00057%) 109. Siliconphthalocyanine bis 5.2 mg/ml Silicon [di(1,6- 0.5 mg/ml 15:1:0:19 THF(70%) 410 798 nm (670 nm) (dimethylhexylvinylsilyloxide)diphenylnaphthalocyanine)] 0.1 mg/ml (0.216 μm CML) (0.00057%)diphthalocyanine bis (dimethylhexylvinylsilyloxide) + Silicon [di(1,6-diphenylnaphthalocyanine)] dinaphthalocyanine bis(dimethylhexylvinylsilyloxide) 110. Silicon phthalocyanine bis 4.8 mg/mlSilicon [di(1,6- 0.5 mg/ml 15:1  THF (70%) 409 798 nm (670 nm)(dimethylhexylvinylsilyloxide) diphenylnaphthalocyanine)] (0.216 μm CML)(0.00057%) dinaphthalocyanine bis (dimethylhexylvinylsilyloxide)

TABLE 2 LOADING CONC. SOLVENT EMISSION HYBRID COMPOUND (mg/ml) SYSTEMLATEX SIZE % SOLID INTENSITY MAXIMUM EXCITATION 1. Silicon [di(1,6- 2.0mg/ml THF 0.216 μm 0.00057% 50 760 nm 650 nm diphenylnaphthalocyanine)]CML diphthalocyanine bis(dimethylhexylvinylsilyloxide) 2. Silicon[di(1,6- 2.0 mg/ml THF 0.216 μm 0.00057% 0 7/0.5 765 nm/825 nm 650 nmdiphenylnaphthalocyanine)] CML tetrafluorophthalocyanine phthalocyaninebis(dimethylhexylvinylsilyloxide) 3. Silicon [di(1,6- 1 5 mg/ml THF0.216 μm 0.00057% 0 5/0 3 770 nm/839 nm 650 nmdiphenylnaphthalocyanine)] CML tetrafluorophthalocyanine phthalocyaninebis(dimethylpentafluorophenylsilyloxide) 4. Silicon [di(1,6- 0.1 mg/mlTHF 0.216 μm 0.00057% 0.2 775 nm 650 nm diphenylnaphthalocyanine)] CMLdiphthalocyanine bis(dimethylpentafluorophenylsyloxide) 5. Silicon[di(1,6- 1.5 mg/ml THF 0.216 μm 0.00057% 7 758 nm 650 nmdiphenylnaphthalocyanine)] di(tert- CML butylphthalocyanine)bis(dimethylhexylvinylsilyloxide) 6. Silicon [di(2,3-naphthalocyanine)]1.0 mg/ml THF 0.216 μm 0.00057% 7 779 nm 650 nmdi(1,4-diphenylphthalocyanine) CML bis(dimethylhexylvinylsilyloxide) 7.Silicon [di(2,3-naphthalocyanine)] 2.0 mg/ml THF 0.216 μm 0.00057% 6 792nm 650 nm di(1,4-diphenylphthalocyanine) CMLbis(dimethylpentalfuorophenylsilyloxide) 8. Silicon[di(1,6-diphenyl-2,3- 2.0 mg/ml THF 0.216 μm 0.00057% 43 757 nm 650 nmnaphthalocyanine)] di(2,3-tert- CML butylphthalocyanine)bis(dimethylhexylvinylsilyloxide) 9. Silicon [di(1,6-diphenyl-2,3- 0.4mg/ml THF 0.216 μm 0.00057% 2 770 nm 660 nm naphthalocyanine)]di(2,3-tert- CML butylphthalocyanine)bis(dimethylpentalfuorophenylsilyloxide) 10. Silicon[di(1,6-diphenyl-2,3- 1.5 mg/ml THF 0.216 μm 0.00057% 58 757 nm 650 nmnaphthalocyanine)] diphthalocyanine CML bis(trihexylisilyloxide) 11.Silicon [di(1,6-diphenyl-2,3- 0.2 mg/ml THF 0.216 μm 0.00057% 15 798 nm350 nm naphthalocyanine)] dinaphthalocyanine CMLbis(dimethylhexylvinylsilyloxide) 12. Silicon (1,6-diphenyl-2,3- 0.8mg/ml THF 0.216 μm 0.00057% 74 720 nm 630 nm naphthalocyanine)]triphthalocyanine CML bis(dimethylhexylvinylsilyloxide) 13. Silicon[di(1,6-diphenyl-2,3- 2.0 mg/ml THF 0.216 μm 0.00057% 34 770 nm 675 nmnaphthalocyanine)] (2,3- CML naphthalocyanine) phthalocyaninebis(dimethylhexylvinylsilyloxide) 14. Silicon [di(2,3-naphthalocyanine)]0.1 mg/ml THF 0.216 μm 0.00057% 1 800 nm 650 nmdi(2,3-dicyanophthalocyanine) CML bis(dimethylhexylvinylsilyloxide) 15.Silicon [di(1,6- 0.5 mg/ml THF 0.216 μm 0.00057% 8 790 nm 650 nmdiphenylnaphthalocyanine)] CML di(dichlorophthalocyanine) 16. Silicon[di(1,6-diphenyl-2,3- 0.5 mg/ml THF 0.216 μm 0.00057% 1 764 nm 660 nmnaphthalocyanine)] diphthalocyanine CML bis[poly(ethylene glycol) methylester] 17. Silicon [di(1,6-diphenyl-2,3- 0.4 mg/ml THF 0.216 μm 0.00057%2 768 nm 670 nm naphthalocyanine)] diphthalocyanine CML dihydroxide 18.Silicon [di(1,6-diphenyl-2,3- 1.0 mg/ml THF 0.216 μm 0.00057% 17 766 nm650 nm naphthalocyanine)] diphthalocyanine CML bis(octyloxide) 19.Silicon [di(1,6-diphenyl-2,3- 0.5 mg/ml THF 0.216 μm 0.00057% 1.0 777 nm660 nm naphthalocyanine)] diphthalocyanine CML bis(phenoxide) 20.Silicon trinaphthalocyanine 0.5 mg/ml THF 0.216 μm 0.00057% 11 782 nm660 nm dichlorophthalocyanine CML bis(dimethylhexylvinylsilyloxide)

EXAMPLE 16 Adsorption of Anti-Human Chorionic Gonadotropin (hCG)Antibody to Latex Particles

A typical example of the adsorptions of an antibody to dyed latexparticles, prepared as described in Example 10, and of a complementaryantibody to undyed latex particles, both of which can be used in asandwich assay for hCG, is outlined below. Those skilled in the art willrecognize that various techniques are available to adsorb or tocovalently couple proteins, peptides, ligand analogues nucleotides andnucleic acids to latex particles. A solution of dye latex (0.1 ml, 2%solids, 412 nm; entry 10, Table 1) was added quickly while vortexing toa solution of anti-β hCG monoclonal antibody (0.2 ml, 6.6 mg/ml; AppliedBiotech Inc., San Diego, Calif.) in 20 mM sodium borate/150 mM sodiumchloride, pH 8.2. A solution of 0.1 M potassium citrate, pH 3, (0.04 ml)was added quickly while vortexing to the antibody latex solution at roomtemperature and the pH of the resulting solution was 3.5. The solutionincubated at room temperature for 5 minutes, then a solution of 2 Mpotassium borate, pH 9.7 (0.025 ml) was added quickly while vortexing tobring the pH to about 8.5. This latex antibody conjugate was dialyzed(Spectra-por dialysis tubing, molecular weight cutoff of 300,000,Spectrum, Houston, Tex.) against 4 changes of 2 L each of 20 mM sodiumborate/150 mM sodium chloride, pH 8.2 at 4° C. for 4 days. The dialyzedlatex conjugate was then removed from the dialysis tubing and the solidsconcentration was calculated to be 0.4%. This conjugate can be used forimmunoassays for hCG in serum. The latex has excitation and emissionwavelengths of 650 nm and 780 nm, respectively.

A solution of polystyrene sulfate latex (0.036 ml, 8.4% solids, 1000 nm;Interfacial Dynamics Corp., Inc., Portland Oreg.) was added quickly, atroom temperature, while vortexing to a solution consisting of anti-α hCGmonoclonal antibody (0.12 ml, 10.3 mg/ml; Applied Biotech Inc. SanDiego, Calif.) in 20 mM sodium borate/150 mM sodium chloride, pH 8.2 and0.1 M potassium citrate, pH 3, (0.6 ml). The solution incubated at roomtemperature for 5 minutes and was subjected to centrifugation in anEppendorf centrifuge (2000×g for 5 min). The supernatant was removed,the pellet was resuspended in 0.1 M potassium phosphate, pH 7, (1.5 ml)and the suspension was subjected to centrifugation as described above.This process was repeated 2 times more and in the final centrifugation,the pellet was resuspended with 0.1 M potassium phosphate, pH 7 (0.3 ml)to make 1% solids. This antibody latex is used on a solid phase, such asa membrane, to capture the hCG-dye antibody latex conjugate complex in areaction mixture in an immunoassay for hCG.

EXAMPLE 17 Immunoassay for hCG

The solid phase anti-α hCG latex solution (0.005 ml, 1% solids; example16) can be applied to a 2 cm² piece of 0.45 micron nylon membrane(Millipore Corp., Boston, Mass.) which has been treated with a 2%solution of condensed milk to lower non-specific binding interactions.This membrane can be used as the solid phase onto which is captured thehCG dye latex conjugate complex. Thus, an hCG assay can be performed byaddition of dye latex conjugate (0.025 ml, example 16) to 0.1 ml samplesof serum suspected of containing hCG and also to 0.1 ml serum samplescontaining known amounts of hCG (10, 100, 300, 500 and 1000 mIU/ml). Theserum samples should be incubated about 10 minutes and then the samplesare applied to the solid phase membrane containing the solid phaselatex. The membrane should be placed over an absorbent so that the serumsample containing the dye latex conjugates flows through the solid phaselatex spot. After the serum solution has passed through the membrane,serum (0.5 ml) not containing the dye latex conjugate is applied to themembrane to remove unbound dye latex conjugate. The latex spots on themembranes are then placed in a front surface fluorescence accessory in afluorometer and the spot is excited at 650 nm and the fluorescenceintensity of the spot on each membrane is measured at 780 nm. Thefluorescence intensity as a function of the hCG concentrations of theknown samples is plotted. The fluorescence intensities of the unknownhCG serum samples can be compared to the known hCG concentrations fromthe graph. The assay protocol of this Example may be performed usingconjugates comprised of water soluble hybrid phthalocyanine derivativesand, for example, proteins, polypeptides, antibodies, nucleic acids andthe like, instead of the dye latex conjugates.

EXAMPLE 18 Fluorometer for Measuring Near Infrared Emitting Dyes

The dye sample (2 ml sample volume in a 10 mm×10 mm quartz cuvette) wasexcited by a diode laser (Sun Laser SL-6; 1=670+/−10 nm, 0.95 mW) whichwas filtered by a low-pass cutoff filter (Corion LS700, passeswavelengths less than 700 nm). Fluorescence emission was detected at 90oto the incident diode laser beam. The emitted light was collected andfocused on a silicon photodiode (Melles Griot, Cat. # 13DS1009) by acondenser consisting of two aspheric lenses (Melles Griot, Cat # 01 LAG119). A high-pass cutoff filter (Schott Glass RG715) in front of theSilicon photodiode blocked scattered laser light at 670 nm but passedemitted light at wavelengths larger than 715 nm. The photocurrent fromthe silicon photodiode was amplified and displayed by a currentamplifier in nanoamps (“nA”), (Melles Griot, Cat. # 13 AMP 003). In someinstances, 12 nm band filters were placed in front of the siliconphotodiode with center wavelengths at 730 nm, 790 nm, 850 nm, and 900nm.

EXAMPLE 19 Synthesis of Silicon 2,3-Naphthalocyaninebis(diphenylvinylsilyl-oxide)

A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05mmol) in dimethylformamide (0.5 ml) containing diphenylvinylchlorosilane(28 μL, 0.125 mmol) and imidazole (7 mg, 0.1 mmol) was stirred underargon at room temperature for 18 hours. The reaction mixture wasevaporated and the residue purified on a silica column equilibratingwith hexane and eluting the product with toluene as a long green band.The toluene fraction containing the product was evaporated to afford 5mg green solid.

EXAMPLE 20 Synthesis of Silicon 2,3-Naphthalocyaninebis(triphenylsilyloxide)

A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05mmol) in dimethylformamide (1 ml) containing triphenylchlorosilane (37mg, 0.125 mmol) and imidazole (7 mg, 0.1 mmol) was stirred under argonat room temperature for 18 hours. The reaction mixture was evaporatedand the residue purified on a silica column equilibrating with hexaneand eluting the product with toluene as a green band. The toluenefraction containing the product was evaporated to afford 2.5 mg greensolid.

EXAMPLE 21 Synthesis of Silicon 2,3-Naphthalocyaninebis(dimethylmaleimidoethoxysilyloxide)

A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05mmol) in dimethylformamide (1 ml) containing dichlorodimethylsilane(13.5 μL, 0.11 mmol) and imidazole (14 mg, 0.2 mmol) was stirred underargon at room temperature for 18 hours. The reaction mixture was thentreated with N-(2-hydroxyethyl)maleimide (35 mg, 0.25 mmol) and stirredfor an additional 10 hours. The reaction mixture was evaporated and theresidue purified on a silica column equilibrating with hexane, thentoluene and eluting the product with toluene/10% isopropanol as a greenband. The eluate containing the product was evaporated to afford 3.5 mgof green solid.

EXAMPLE 22 Synthesis of Silicon 2,3-Naphthalocyaninebis(dimethylsilyloxide-trans-stilbene)

A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05mmol) in dimethylformamide (1 ml) containing dichlorodimethylsilane(13.5 μL, 0.11 mmol) and imidazole (14 mg, 0.2 mmol) was stirred underargon at room temperature for 2 hours. The reaction mixture was thentreated with trans-4-hydroxystilbene (49 mg, 0.25 mmol) and stirred foran additional 5 hours. The reaction mixture was evaporated and theresidue purified on a silica column equilibrating with hexane andeluting the product with toluene as a long green band. The toluenefraction containing the product was evaporated to afford 4 mg greensolid.

EXAMPLE 23 Synthesis of Silicon 2,3-Naphthalocyaninebis(dimethylhexylvinyl-silyloxide

A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05mmol) in dimethylformamide (1 ml) containing7-oct-1-enyldimethylchlorosilane (32 μL, 0.125 mmol) and imidazole (7mg, 0.1 mmol) was stirred under argon at room temperature for 18 hours.The reaction mixture was evaporated and the residue purified on silicacolumn equilibrating with hexane and eluting the product with toluene asa green band. The toluene fraction containing the product was evaporatedand the residue treated with hexane to afford a dark green solid andlight green supernatant. The mixture was centrifuged, the supernatantremoved and the solid treated with more hexane and centrifuged. Thesupernatant was again removed and the solid dried under vacuum to yield7.3 mg of product.

EXAMPLE 24 Synthesis of Silicon 2,3-Naphthalocyaninebis(tridecafluoro-1,1,-2,2-tetrahydrooctyl-1-dimethylsilyloxide)

A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05mmol) in dimethylformamide (1 ml) containing(tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-dimethylchlorosilane (37 μL,0.1 mmol) and imidazole (7 mg, 0.1 mmol) was stirred under argon at roomtemperature for 2 hours. The reaction mixture was evaporated and theresidue purified on a silica column equilibrating with hexane andeluting with hexane/20% toluene followed by hexane/40% toluene to affordthe product as a green band. The product eluate was evaporated and theresidue treated with hexane to afford a green solid. The mixture wascentrifuged, the supernatant removed and the solid treated with morehexane and recentrifuged. The supernatant was again removed and thegreen solid dried under vacuum to yield 7.5 mg of product.

EXAMPLE 25 Synthesis of Silicon 2,3-Naphthalocyaninebis(dimethylretinol)

A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05mmol) in dimethylformamide (1 ml) containing dichlorodimethylsilane(13.5 μL, 0.11 mmol) and imidazole (14 mg, 0.2 mmol) was stirred underargon at room temperature. After 20 minutes, the reaction mixture wastreated with all-trans-retinol (72 mg, 0.25 mmol) and stirred for anadditional 1 hour. The reaction mixture was evaporated and the residuepurified on a silica column equilibrating with hexane and eluting theproduct with toluene as a long green band. The toluene fractioncontaining the product was evaporated and the residue treated withhexane to yield a dark green solid and light green supernatant. Themixture was centrifuged, the hexane removed and the solid dried undervacuum to yield 10 mg of final product.

EXAMPLE 26 Synthesis of Silicon (IV)5,9,14,18,23,27,32,36-octaethoxy-2,3-naphthalocyanine dichloride(abbreviated as: Silicon octaethoxy-2,3-naphthalocyanine dichloride))

4,9-Diethoxy-1,3-diiminobenz[f]isoindoline (0.6 g) was added under argonto freshly distilled quinoline (12 ml). After stirring for 10 minutes,silicon tetrachloride (4.0 ml) was added and the reaction mixture washeated at 190° C. for 1 hour. The reaction mixture was cooled to roomtemperature, and water (120 ml) was added slowly to hydrolyze theunreacted silicon tetrachloride. The blue-black precipitate was filteredoff and washed sequentially with methanol (5 ml) and acetone (5 ml).

UV-vis (methylene chloride) (λ_(max)(nm)): 768, 869.

EXAMPLE 27 Synthesis of Silicon (IV)5,9,14,18,23,27,32,36-octaethoxy-2,3-naphthalocyanine dihydroxide(abbreviated as: Silicon octaethoxy-2,3-naphthalocyanine dihydroxide)

A suspension of silicon octaethoxy-2,3-naphthalene dichloride (1.96 g)in pyridine (15 ml) containing water (15 ml) was refluxed for 18 hours.The suspension was cooled, the black precipitate filtered and washedwith water (10 ml). The precipitate was dried under vacuum and weighed(1.37 g, purple powder).

UV-vis (methylene chloride) (λ_(max)(nm)): 766, 867.

EXAMPLE 28 Synthesis of Silicon (IV)5,9,14,18,27,32,36-octaethoxy-2,3-naphthalocyanine bis(7-oct-1-enyldimethyl silyloxide) (abbreviated as: Siliconoctaethoxy-2,3-naphthalocyanine bis(dimethylhexylvinylsilyloxide

A suspension of silicon IV octaethoxy-2,3-naphthalocyanine dihydroxide(1.0 g) in dimethylformamide (20 ml) containing7-oct-1-enyldimethylchlorosilane (0.6 ml) and imidazole (140 mg) wasstirred under argon at room temperature for 24 hours. The reactionmixture was evaporated with a rotary evaporator, chromatographed on asilica gel (70-230 mesh, 60 Å,) column (2×50 cm) equilibrated in hexane.The product was eluted sequentially with hexane and hexane-toluene(1:1)), vacuum dried, and weighed (46 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm), ε (M⁻¹cm⁻¹)): 855, 370000.

Infrared Spectrum(KBr): 3074, 2958, 2924, 2854, 1589, 1417, 1373, 1348,1262, 1238, 1194, 1161, 1111, 1044, 1025, 933, 909, 844, 799, 760 cm⁻¹.

¹H-NMR (500 MHz, CDCl₃): δ 9.0 (m, 2,5-Nc), 7.9 (m, 3,4-Nc), 5,3 (m,—CH₂), 4.6 (m, vinyl —CH₂), 3.5 (m, vinyl CH), 1.8 (m, —CH₃), 1.3 (m, ε—CR₂), 0.5 (m, δ CH₂), 0.1 (m, γ —CH₂), −0.8 (m, β —CH₂), −1.7 (m, α—CH₂), −2.3 (s, —CH₃).

EXAMPLE 29 Synthesis of Silicon Phthalocyaninebis(dimethylmaleimido-fluorescein)

Fluorescein ATP (0.5 mg, 1.05 mmol) was treated with a solution of 0.12M potassium carbonate in 80% methanol (52 μL). After 5 minutes, thehydrolysis solution was quenched by the addition of 0.5 M potassiumphosphate/0.1 M potassium borate, pH 7.0 in 1 N HCl (10 μL). Thequenched hydrolysis solution was evaporated to dryness, redissolved indimethylformamide (100 μL) and the resulting solution added to siliconphthalocyanine bis(dimethylmaleimidosilyloxide) in a 1.0 ml serum vial.The reaction mixture was then stirred at room temperature for 1 hour.The crude product was then chromatographed on two 3″×3″ silica platesusing toluene/20% dimethylformamide. After elution, the plates weredried under vacuum and rechromatographed for a better separation. Theproduct band was scraped off, and treated with dimethylformamide (5 ml),vortexed 30 seconds and filtered from the silica. The filtrates wereevaporated to give 0.55 mg of greenish fluorescent solid.

EXAMPLE 30 Synthesis of Tin (IV)5,9,14,18,23,27,32,36-octabutoxy-2,3-naphthalocyaninebis(triethylsilyloxide))

A mixture of triethylsilanol (77 μL), sodium (3.5 mg), and xylenes (5ml) was refluxed under argon for 1 hour. A solution of Tin (IV)octabutoxy-2,3-naphthalocyanine dichloride (74 mg) in xylenes (5 ml) wasadded to the solution formed and the mixture was refluxed for 20minutes. The resultant was washed twice with water (25 ml each time),dried (MgSO₄), and evaporated to a dark red solid with a rotaryevaporator. This solid was chromatographed on a silica gel (70-230 mesh,60 Å,) column (2×50 cm) equilibrated in hexane and eluted sequentiallywith toluene and toluene-10% isopropanol. The product was vacuum dried,and weighed (17 mg).

UV-vis(tetrahydrofuran) (λ_(max)(nm)), ε (M⁻¹cm⁻¹)): 900, 174000.

EXAMPLE 31 Synthesis of Tin (IV) 2,3-naphthalocyaninebis(triethylsilyloxide)

A mixture of triethylsilanol (77 μL), sodium (3.5 mg), and xylenes (8ml) was refluxed under argon for 1 hour. Tin (IV) 2,3-naphthalocyaninedichloride (45 mg) was added to the solution formed, and the mixture wasrefluxed for 5 days. The suspension was filtered, and the solid waswashed sequentially with xylenes and water, vacuum dried, and weighed(41 mg). The solid was chromatographed on a silica gel (70-230 mesh, 60Å,) column (2×50 cm) equilibrated with methylene chloride and elutedsequentially with methylene chloride—20% tetrahydrofuran, methylenechloride—50% tetrahydrofuran and finally tetrahydrofuran. The productwas triturated with hexane (2 ml), vacuum dried, and weighed (26 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)), ε (M⁻¹cm⁻¹)): 700; 746; 786,253000.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 820.

EXAMPLE 32 Synthesis of Tin (IV) 2,3-naphthalocyaninebis(7-oct-1-enyldimethylsilyloxide) (abbreviated as: Tin (IV)2,3-naphthalocyanine bis(dimethylhexylvinylsilyloxide))

A mixture of 7-oct-1-enyldimethylsilanol (186 mg), sodium (7 mg), andxylenes (10 ml) was refluxed under argon for 4 hours. Tin (IV)2,3-naphthalocyanine dichloride (90 mg) was added to the solution formedand the mixture was refluxed for 4 days. The suspension was filtered andthe solid was washed sequentially with xylenes (5 ml) and water (5 ml).The organic layer of the filtrate was separated, dried (MgSO₄), andevaporated with a rotary evaporator. The residue was triturated twicewith hexane (2 ml each time) to afford a bright green solid which wasvacuum dried and weighed (8.5 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm), ε (M⁻¹cm⁻¹)): 670, 7200; 732,69900; 786, 84900.

EXAMPLE 33 Synthesis of Tin (IV)5,9,14,18,23,27,32,36-octabutoxy-2,3-naphthalocyanine dichloride

Tin tetrachloride (234 μL) was added to a mixture ofoctabutoxy-2,3-naphthalocyanine (310 mg) in dry dimethylformamide (15ml) under an argon atmosphere and the mixture refluxed with stirring for6 hours. The resultant was allowed to cool, the suspension was filtered,and the dark red solid was washed sequentially with dimethylformamide (5ml) and water (5 ml), vacuum dried and weighed (288 mg).

EXAMPLE 34 Synthesis of Tin (IV)5,9,14,18,23,27,32,36-octabutoxy-2,3-naphthalocyaninebis(7-oct-1-enyldimethylsilyloxide (abbreviated as: Tin (IV)Octabutoxy-2,3-naphthalocyanine bis(dimethylhexylvinylsilyloxide))

A mixture of 7-oct-1-enyldimethylsilanol (186 mg), sodium (7 mg), andxylenes (10 ml) was refluxed under argon for 5 hours. Tin (IV)octabutoxy-2,3-naphthalocyanine dichloride (37 mg) was added to thesolution formed, and the mixture was refluxed for 2 days. The resultantwas washed with water (10 ml), dried (MgSO₄), and evaporated to a darkred solid with a rotary evaporator. This solid was chromatographed on asilica gel (70-230 mesh, 60 Å,) column (2×50 cm) equilibrated in hexaneand eluted sequentially with toluene and toluene—10% isopropanol. Theproduct was vacuum dried, and weighed (17 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm), ε (M⁻¹ cm⁻¹)): 785; 893, 227000.

Fluorescence(tetrahydrofuran) (λ_(max)(nm)): 789.

EXAMPLE 35 Synthesis of 7-oct-1-enyldimethylsilanol

A solution of 7-oct-1-enyldimethylchlorosilane (2.56 ml) in ether (2 ml)was added dropwise over 1 hour to a stirring mixture of triethylamine(1.5 ml), water (0.18 ml) and ether (15 ml) in an ice/water bath. Theresultant was stirred a further 1 hour in the ice/water bath andfiltered washing the filtered solid with ether (10 ml). The filtrate wasevaporated with a rotary evaporator and the residue partitioned betweenhexane (30 ml) and water (30 ml). The organic layer was separated, dried(MgSO₄) and filtered through silica gel (70-230 mesh, 60 Å), washingwith hexane (100 ml). The filtrate was evaporated with a rotaryevaporator to afford a colorless oil which was vacuum dried add weighed(1.06 g).

EXAMPLE 36 Synthesis of2,3,20,21-tetrabromo)-9,14,27,32-tetrabutoxy-2,3-naphthalocyanine

1,4-dibutoxynaphthalene-2,3-dicarbonitrile (161 mg) and2,3-dibromo-6,7-dicyanonaphthalene (168 mg) were added to a refluxingsolution of lithium metal (35 mg) in 1-butanol (2 ml) under an argonatmosphere. The reaction solution was maintained at reflux for 2 hours,cooled, and stirred into glacial acetic acid (10 ml). After 30 minutes,the solvent was evaporated with a rotary evaporator and the residuedissolved in methylene chloride (10 ml). The solution was washed twicewith 1 N hydrochloric acid (10 ml each time), followed by water (10 ml),dried (MgSO₄) and evaporated with a rotary evaporator. The residue waschromatographed on a silica gel (70-230 mesh, 60 Å,) column (2×50 cm)equilibrated in hexane and eluted sequentially with hexane—10% toluene,hexane—20% toluene, hexane—30% toluene, hexane—40% toluene and finallyhexane—50% toluene. The solid product was triturated with hexane (2 ml),vacuum dried, and weighed (8 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 743, 839.

Fluorescence(tetrahydrofuran) (λ_(max)(nm)): 789.

EXAMPLE 37 Synthesis of2¹,2⁶,7¹,7⁶/12¹,12⁶-tetrabutoxydinaphtho[b,g/l]-7,12/17-octafluorodibenzo[g,l/q]-5,10,15,20-tetraazoporphyrin(abbreviated as: Di(1,6-dibutoxy-2,3-naphthalocyanine)di(tetrafluorophthalocyanine)

1,4-Dibutoxynaphthalene-2,3-dicarbonitrile (161 mg) andtetrafluorophthalonitrile (100 mg) were added to a refluxing solution oflithium metal (35 mg) in 1-butanol (2 ml) under an argon atmosphere. Thereaction solution was maintained at reflux for 1 hour, cooled, andstirred into glacial acetic acid (10 ml). After 30 minutes the solventwas evaporated with a rotary evaporator and the residue dissolved inmethylene chloride (10 ml). The solution was washed twice with 1 Nhydrochloric acid (10 ml each time), followed by water (10 ml), dried(MgSO₄) and evaporated with a rotary evaporator. The residue waschromatographed twice on a silica gel (70-230 mesh, 60 Å, 2×50cm),column equilibrated in hexane and eluted sequentially withhexane—10% toluene, hexane—20% toluene, hexane—30% toluene, and finallyhexane—40% toluene. The product was vacuum dried and weighed (10 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm), ε (M⁻¹cm⁻¹)): 679, 25800; 752,88200; 789, 76500.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 815.

EXAMPLE 38 Synthesis of2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,7-octafluorodibenzo[g,q]-5,10,15,20-tetraazoporphyrin(abbreviated as: Di(1,6-diphenyl-2,3-naphthalocyanine)di(tetrafluorophthalocyanine))

1,4-diphenylnaphthalene-2,3-dicarbonitrile (165 mg) andtetrafluorophthalonitrile (100 mg) were added to a refluxing solution oflithium metal (35 mg) in 1-butanol (2 ml) under an argon atmosphere. Thereaction solution was maintained at reflux for 1.5 hours, cooled, andstirred into glacial acetic acid (10 ml). After 30 minutes, the solventwas evaporated with a rotary evaporator and the residue dissolved inmethylene chloride (10 ml). The solution was washed twice with 1 Nhydrochloric acid (10 ml each time), followed by water (10 ml), dried(MgSO₄), and evaporated with a rotary evaporator. The residue waschromatographed on a silica gel (70-230 mesh, 60 Å,) column (2×50 cm)equilibrated in hexane and eluted sequentially with hexane—10% toluene,hexane—20% toluene, hexane—30% toluene, hexane—40% toluene and finallyhexane—50% toluene. The bright green product was vacuum dried andweighed (7 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm), ε (M⁻¹cm⁻¹)): 747, 86800.

Fluorescence(tetrahydrofuran) (λ_(max)(nm)): 760.

EXAMPLE 39 Synthesis of Dibutoxy-1,3-diiminobenz[f]isoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture of1,4-dibutoxynaphthalene-2,3-dicarbonitrile (1.61 g), 25% sodiummethoxide in methanol (1.14 ml), and dry 1-butanol (10 ml) for 30minutes. With continued ammonia introduction, the mixture was refluxedfor 30 minutes. After the resultant had cooled, the solvent was removedunder vacuum with a rotary evaporator. The residue was chromatographedon a silica gel (70-230 mesh, 60 Å,) column (2×50 cm), equilibrated inhexane and eluted sequentially with toluene, toluene—1% isopropanol,toluene—2% isopropanol, toluene—5% isopropanol, toluene—10% isopropanoland finally toluene—20% isopropanol. The yellow product was treated withether (10 ml), collected by filtration, washed with ether (10 ml),vacuum dried and weighed (517 mg).

¹H-NMR (500 MHZ, CDCl₃) δ 8.22 (m, 5,8-H), 7.65 (m, 6,7-H), 4.23 (m, γ—CH₂), 1.97 (m, β —CH₂), 1.61 (m, α —CH₂), 1.04 (t, —CH₃).

EXAMPLE 40 Synthesis of 4,9-diethoxy-1,3-diiminobenz[f]isoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture of1,4-diethoxynaphthalene-2,3-dicarbonitrile (1.33 g), 25% sodiummethoxide in methanol (1.14 ml), and dry ethanol (10 ml) for 20 minutes.With continued ammonia introduction, the mixture was refluxed for 2hours. After the resultant had cooled, the solvent was removed undervacuum with a rotary evaporator. The residue was treated with methylenechloride (10 ml) and the product was collected by filtration, washedsequentially with water (5 ml) and methylene chloride (5 ml), vacuumdried and weighed (766 mg).

EXAMPLE 41 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]silicondihydroxide (abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)]diphthalocyaninedihydroxide)

Silicon tetrachloride (231 μL) was added to a mixture ofdiphenyl-1,3-diiminobenz[f]isoindoline (470 mg) and1,3-diiminoisoindoline (97 mg) in freshly distilled quinoline (5 ml)under an argon atmosphere and the mixture heated with stirring at 200°C. for 40 minutes. The resultant was allowed to cool to 160° C., treatedwith water (5 ml) and refluxed for 5 minutes. The mixture was cooled,treated with ether (30 ml) and filtered washing the solid sequentiallywith ether (10 ml) and water (10 ml). The organic layer of the filtrate(which was dark green) was separated from the aqueous layer, washed withwater (15 ml), dried (MgSO₄) and evaporated with a rotary evaporator.The residue was chromatographed three times on a silica gel (70-230mesh, 60 Å,) column (2×50 cm) equilibrated in hexane and elutedsequentially with hexane, hexane—10% methylene chloride, hexane—20%methylene chloride, and finally hexane—50% methylene chloride. Theproduct was vacuum dried and weighed (55.5 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm), ε (M⁻¹ cm⁻¹): 640; 680; 714,67900; 742.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 750.

EXAMPLE 42 Synthesis of[2¹,2⁶,7¹,7⁶/12¹,12⁶-tetraethoxydinaphtho[b,g/l]-7,12/17-dibenzo[g,l/q]-5,10,15,20-tetraazoporphorinato]silicondihydroxide] (abbreviated as:Silicon[di(1,6-diethoxy-2,3-naphthalocyanine)]diphthalocyaninedihydroxide)

Silicon tetrachloride (137 μL) was added to a mixture of4,9-diethoxy-1,3-diiminobenz[f]isoindoline (227 mg) and1,3-diiminoisoindoline (58 mg) in freshly distilled quinoline (3 ml)under an argon atmosphere and the mixture heated with stirring at 200°C. for two hours. The resultant was allowed to cool 160° C., treatedwith water (3 ml) and refluxed for 5 minutes. The mixture was cooled,treated with ether (10 ml), and the dark blue solid product filteredoff, washing the solid sequentially with ether (10 ml) and water (10ml), vacuum dried and weighed (175 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 600, 632, 666, 700, 724, 788.

EXAMPLE 43 Synthesis of[2¹,2⁶,7¹,7⁶/12¹,12⁶-tetraethoxydinaphtho[b,g/l]-7,12//17-dibenzo[g,l/q]-5,10,15,20-tetraazoporphyrinato]siliconbis(7-oct-1-enyldimethylsilyloxide (abbreviated as:Silicon[di(1,6-diethoxy-2,3-naphthalocyanine)]diphthalocyaninebis(dimethylhexylvinylsilyloxide))

A mixture of silicon(di(1,6-diethoxy-2,3-naphthalocyanine)]diphthalocyanine dihydroxide (85mg), 7-oct-1-enyldimethylchlorosilane (256 μL), imidazole (68 mg), anddimethylformamide (2 ml) was stirred at room temperature for 24 hours.The resultant was concentrated under vacuum with a rotary evaporator.The residue was chromatographed on a silica gel (70-230 mesh, 60 Å,)column (2×50 cm) equilibrated in hexane and eluted sequentially withtoluene and toluene—1% isopropanol. The product was vacuum dried andweighed (32 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 601, 633, 667, 702, 731, 822,904.

EXAMPLE 44 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(7-oct-1-enyldimethylsilyloxide)(abbreviated as: Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)])diphthalocyaninebis(dimethylhexylvinylsilyloxide) (FIG. 9)

A mixture ofsilicon[di(1,6-diphenyl-2,3-naphthalocyanine)]diphthalocyaninedihydroxide (30 mg), 7-oct-1-enyldimethylchlorosilane (115 μL),imidazole (30 mg) and dimethylformamide (650 μL) was stirred at roomtemperature for 30 minutes. The resultant was concentrated under vacuumon the rotary evaporator. The residue was chromatographed on a silicagel (70-230 mesh, 60 Å,) column (2×50 cm) equilibrated in hexane andeluted sequentially with hexane and toluene. The product was vacuumdried and weighed (38 mg).

¹H-NMR (500 MHZ, CDCl₃) δ 8.31, 8.25 (m, 2,5-Nc, 10,13-Nc), 7.94 (m,Ar-Nc), 7.95, 7.74 (3,4-Nc, 11,12-Pc), 0.68 (m, ε —CH₂), 0.21 (m, δ—CH₂), −0.11 (m, γ —CH₂), −1.22 (m, β —CH₂), −2.14 (m, α —CH₂), −2.76(s, —CH₃).

UV-vis(tetrahydrofuran) (λ_(max)(nm), ε (M⁻¹ cm⁻¹)): 644; 684; 718,81100; 748.

Fluorescence(tetrahydrofuran) (λ_(max)(nm)): 754.

EXAMPLE 45 Synthesis of Tetrafluoro-1,3-diiminobenz[f]isoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture oftetrafluorophthalonitrile (2.0 g), 25% sodium methoxide in methanol (2.3ml), and dry 1-butanol (10 ml) for 20 minutes. With continued ammoniaintroduction, the mixture was refluxed for 1 hour. After the resultanthad cooled, the solvent was removed under vacuum with a rotaryevaporator. The residue was treated with ether (50 ml) and the productwas collected by filtration, washed sequentially with water (10 ml), andether (10 ml), vacuum dried and weighed (0.45 g).

EXAMPLE 46 Synthesis of 4,7-diphenyl-1,3-diiminobenz[f]isoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture of1,4-diphenylnaphthalene-2,3-dicarbonitrile (4.3 g), 25% sodium methoxidein methanol (3.0 ml), and dry 1-butanol (25 ml) for 30 minutes. Withcontinued ammonia introduction, the mixture was refluxed for 1.5 hours.After the resultant had cooled, the solvent was removed under vacuumwith a rotary evaporator. The residue was treated with methylenechloride (50 ml) and the product was collected by filtration, washedsequentially with water (10 ml) and methylene chloride (10 ml), vacuumdried and weighed (3.68 g).

EXAMPLE 47 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-octafluorodibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]silicondihydroxide (abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)1di(tetrafluorophthalocyanine)dihydroxide)

Silicon tetrachloride (86 μL) was added to a mixture ofdiphenyl-1,3-diiminobenz[f]isoindoline (174 mg) andtetrafluoro-1,3-diiminoisoindoline (54 mg) in freshly distilledquinoline (1 ml) under an argon atmosphere and the mixture heated withstirring at 200° C. for 1 hour. The resultant was allowed to cool to160° C., treated with water (1 ml) and refluxed for 5 minutes. Themixture was cooled, treated with ether (10 ml) and filtered washing thesolid sequentially with water (2 ml) and ether (5 ml). The organic layerof the filtrate was separated, washed with water (5 ml), dried (MgSO₄)and evaporated with a rotary evaporator. The residue was chromatographedon a silica gel (70-230 mesh, 60 Å, 2×50 cm) column equilibrated inmethylene chloride and eluted with methylene chloride. The product wasvacuum dried and weighed (18 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 727, 759, 809, 835.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 685, 760, 840.

EXAMPLE 48 Synthesis of [2¹,2²,12¹,12⁶-tetraphenyldinaphtho[b,l]-71,76diethoxynaphtho[g]-17-benzo[q]-5,10,15,20-tetraazoporphyrinato]silicondihydroxide (abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)])1,6-diethoxyphthalocyanine)phthalocyaninedihydroxide)

Silicon tetrachloride (172 μL) was added to a mixture ofdiphenyl-1,3-diiminobenz[f]isoindoline (347 mg),diethoxy-1,3-diiminobenz[f]isoindoline (71 mg) and1,3-diiminoisoindoline (36 mg) in freshly distilled quinoline (2 ml)under an argon atmosphere and the mixture heated with stirring at 200°C. for 1 hour. The resultant was allowed to cool to 160° C., treatedwith water (2 ml) and refluxed for 5 minutes. The mixture was cooled,treated with ether (10 ml) and filtered washing the solid sequentiallywith water (5 ml) and ether (5 ml). The organic layer of the filtratewas separated, washed with water (10 ml), dried (MgSO₄) and evaporatedwith a rotary evaporator. The residue was chromatographed on a silicagel (70-230 mesh, 60 Å, 2×50 cm) column equilibrated in methylenechloride and eluted with methylene chloride. The product was vacuumdried and weighed (6 mg).

UV-vis (methylene chloride) (λ_(max)(nm)): 649, 693, 724, 758, 827.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 750.

EXAMPLE 49 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7-tetrafluoronaphtho[g-17-benzo[q]5,10,15,20-tetraazoporphyrinato]silicondihydroxide (abbreviated as: Silicondi(1,6-diphenyl-2,3-naphthalocyanine)](tetrafluorophthalocyanine)phthalocyaninedihydroxide)

Silicon tetrachloride (172 μL) was added to a mixture ofdiphenyl-1,3-diiminobenz[f]isoindoline (347 mg),tetrafluoro-1,3-diiminobenz[f]isoindoline (54 mg) and1,3-diiminoisoindoline (36 mg) in freshly distilled quinoline (2 ml)under an argon atmosphere and the mixture heated with stirring at 200°C. for 1 hour. The resultant was allowed to cool to 160° C., treatedwith water (2 ml) and refluxed for 5 minutes. The mixture was cooled,treated with ether (10 ml) and filtered washing the solid sequentiallywith water (5 ml) and ether (5 ml). The organic layer of the filtratewas separated, washed with water (10 ml), dried (MgSO₄) and evaporatedwith a rotary evaporator. The residue was chromatographed on a silicagel (70-230 mesh, 60 Å, 2×50 cm) column equilibrated in methylenechloride and eluted with methylene chloride. The product was vacuumdried and weighed (21 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 646, 689, 720, 753, 790.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 760.

EXAMPLE 50 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]7-tetrafluoronaphtho[1-17-5,10,15,20-tetraazoporphyrinato]siliconbis(7-oct-1-enyldimethylsilyloxide (abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)](tetrafluorophthalocyanine)phthalocyaninebis(dimethylhexylvinylsilyloxide))

A mixture ofsilicon[di(1,6-diphenyl-2,3-naphthalocyanine)](tetrafluorophthalocyanine)phthalocyaninedihydroxide (10.5 mg), 7-oct-1-enyl dimethylchlorosilane (38 μL),imidazole (10 mg) and dimethylformamide (200 μL) was stirred at roomtemperature for 30 minutes. The resultant was concentrated under vacuumon the rotary evaporator. The residue was chromatographed on a silicagel (70-230 mesh, 60 Å, 2×50 cm) column equilibrated in hexane andelated with toluene. The product was vacuum dried and weighed (4 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 732, 757, 794, 816.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 763, 830.

EXAMPLE 51 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7-tetrafluoronaphtho[g]-17-benzo[q]-5,10,15,20-tetraazoporphyrinato]siliconbis(dimethylpentafluoro phenylsilyloxide) (abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)](tetrafluorophthalocyanine)phthalocyaninebis(dimethylpentafluorophenylsilyioxide))

A mixture ofsilicon[di(1,6-diphenyl-2,3-naphthalocyanine)](tetrafluorophthalocyanine)phthalocyaninedihydroxide (10.5 mg), chlorodimethylpentafluorophenylsilane (28 μL),imidazole (10 mg) and dimethylformamide (200 μL) was stirred at roomtemperature for 30 minutes. The resultant was concentrated under vacuumon the rotary evaporator. The residue was chromatographed on a silicagel (70-230 mesh, 60 Å, 2×50 cm) column equilibrated in hexane andeluted with hexane—50% toluene to afford two product fractions A and Bwhich were vacuum dried and weighed (2.8 mg and 5.5 mg, respectively).

A. UV-vis (tetrahydrofuran) (λ_(max)(nm)): 650, 726, 762, 796, 824.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 770.

B. UV-vis (tetrahydrofuran) (λ_(max)(nm)): 651, 726, 763, 796, 824.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 770.

EXAMPLE 52 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(dimethylpentafluorophenylsilyloxide) (abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)]diphthalocyaninebi(dimethylpentafluorophenylsilyioxide)

A mixture of silicondi(1,6-diphenyl-2,3-naphthalocyanine)]diphthalocyanine dihydroxide (20mg), chlorodimethylpentafluorophenylsilane (58 μL), imidazole (20 mg)and dimethylformamide (450 μL) was stirred at room temperature for 1hour. The resultant was concentrated under vacuum on the rotaryevaporator. The residue was treated with hexane (5 ml) and the greensolid product collected by filtration, washed with hexane (2 ml), vacuumdried and weighed (26 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 648, 691, 724, 759.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 768.

EXAMPLE 53 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraaza(21H),(23H), porphyrin (abbreviated as:di(1,6-diphenyl-2,3-naphthalocyanine)di(2,3-tert-butylphthalocyanine)

A mixture of 1,4-diphenylnaphthalene dicarbonitrile (495 mg),4-tert-butylphthalonitrile (92 mg), and lithium butoxide (4.0 ml) wasrefluxed in an oil bath for 1.5 hours and cooled. Cold glacial aceticacid (20 ml) was added to the suspension formed and vacuum dried. Thegreen residue was resuspended in dichloromethane and the solutioncentrifuged at 3000 rpm for 15 minutes. The supernatant was washed with1 N HCL (2×20 ml) followed by water (1×10 ml). The organic layer wasdried under vacuum. The crude product was chromatographed on a silicagel (70-230 mesh, 60 Å, 2×50 cm) column equilibrated in hexane. Theproduct was eluted sequentially with hexane and toluene, vacuum driedand weighed (4.2 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm), ε (M⁻¹cm⁻¹)): 668, 43297; 688,86914; 726, 92715; 758, 64329.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 732.

EXAMPLE 54 Synthesis of 5-tert-butyl-1,3-diiminoisoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture of4-tert-butylphthalonitrile (1.8 g), 25% sodium methoxide in methanol(2.3 ml), and dry 1-pentanol (20 ml) for 30 minutes. With continuedammonia introduction, the mixture was refluxed for 1.5 hours. After theresultant had cooled, the solvent was removed with a rotary evaporator.The residue was treated with methylene chloride (20 ml) and the productwas collected by filtration, washed sequentially with methylene chloride(20 ml), ether (10 ml), vacuum dried and weighed (0.4 g).

EXAMPLE 55 Synthesis of 6,7-dibromo-1,3-diiminobenz[f]isoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture of6,7-dibromonaphthalene-2,3-dicarbonitrile (0.5 g), 25% sodium methoxidein methanol (0.3 ml), and dry 1-pentanol (10 ml) for 50 minutes. Withcontinued ammonia introduction, the mixture was refluxed for 2.5 hours.After the resultant had cooled, the orange-yellow solid was collected byfiltration and washed with ether (20 ml), vacuum dried and weighed (0.6g).

EXAMPLE 56 Synthesis of Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)di-tert-butylphthalocyanine]dihydroxide

Silicon tetrachloride (57 μL) was added to a mixture ofdiphenyl-1,3-diiminobenz[f]isoindoline (172 mg) and5-tert-butyl-1,3-diiminoisoindoline (50 mg) in freshly distilledquinoline (1 ml) under an argon atmosphere and the mixture heated withstirring at 210° C. for 1 hour. The resultant was allowed to cool,treated with water (2 ml) and refluxed for 5 minutes. The mixture wascooled, treated with ether (10 ml) and filtered washing the solid withether (30 ml). The organic layer of the filtrate was separated, washedtwice with water (20 ml each time), dried (Na₂SO₄) and the etherevaporated with a rotary evaporator. The residue was chromatographed ona silica gel (70-230 mesh, 60 Å, 2×50 cm), column equilibrated withhexane. The product was eluted with methylene chloride, vacuum dried andweighed (11 mg, green solid).

UV-vis (methylene chloride) (λ_(max)(nm)): 656, 670, 694, 730, 758.

Fluorescence (methylene chloride) (λ_(max)(nm)): 767.

EXAMPLE 57 Synthesis ofSilicon[di(1,6-diphenyl-2,3-naphthalocyanine)]di-tert-butylphthalocyaninebi(dimethylhexylvinylsilyloxide)

A mixture ofsilicon[di(1,6-diphenyl-2,3-naphthalocyanine)]di(2,3-tert-butylphthalocyanine)dihydroxide(320 mg), 7-oct-1-enyldimethylchlorosilane (200 μL), imidazole (136 mg)and dimethylformamide (6 ml) was stirred at room temperature for 12hours. The resultant was concentrated under vacuum on the rotaryevaporator. The residue was chromatographed on a silica gel (70-230mesh, 60 Å, 2×50 cm) column equilibrated and eluted with hexane. Theblue product was vacuum dried and weighed (150 mg).

UV-vis (methylene chloride) (λ_(max)(nm)): 632, 676, 702, 750.

Fluorescence (methylene chloride) (λ_(max)(nm)): 716.

EXAMPLE 58 Synthesis of Silicon (Iv)2,3,11,12,20,21,29,30-octabromo-2,3-naphthalocyanine dihydroxide(abbreviated as: silicon octabromo-2,3-naphthalocyanine dihydroxide)

Silicon tetrachloride (114 μL) was added to a mixture of6,7-dibromo-1,3-diiminobenz[f]isoindoline (433 mg) and5-tert-butyl-1,3-diiminoisoindoline (100 mg) in freshly distilledquinoline (2 ml) under an argon atmosphere and the mixture heated withstirring at 210° C. for 2 hours. The resultant was allowed to cool,treated with water (2 ml) and refluxed for 15 minutes. The mixture wascooled, treated with ether (4 ml) and filtered washing the solid threetimes with ether (2 ml each time). The solid was vacuum dried andweighed (0.57 g, dark green solid).

EXAMPLE 59 Synthesis of Silicon (Iv)2,3,11,12,20,21,29,30-octabromo-2,3-naphthalocyaninebis(7-oct-1-enyldimethyl silyloxide) (abbreviated as: siliconoctabromo-2,3-naphthalocyanine bis(dimethylhexylvinylsilyloxide))

A mixture of silicon octabromo-2,3-naphthalocyanine dihydroxide (500mg), 7-oct-1-enyl dimethylchlorosilane (256 μL), imidazole (68 mg) anddimethylformamide (5 ml) was stirred at room temperature for 12 hours.The resultant was concentrated under vacuum with a rotary evaporator.The residue was chromatographed on a silica gel (70-230 mesh, 60 Å, 2×50cm) column equilibrated in hexane. The product was eluted with toluene,vacuum dried and weighed (300 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 694, 702 sh.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 706.

EXAMPLE 60 Synthesis of Silicon (IV)1,4,8,11,15,18,22,25-octaethoxyphthalocyanine dichloride (abbreviatedas: silicon octaethoxyphthalocyanine dichloride)

Silicon tetrachloride (600 μL) was added to a mixture of4,7-diethoxy-1,3-diiminoisoindoline (1.0 g) in freshly distilledquinoline (10 ml) under an argon atmosphere and the mixture heated withstirring at 200° C. for 1.5 hours. The resultant was allowed to cool andtreated with water (10 ml) followed by methylene chloride (10 ml). Theorganic layer was separated and evaporated with a rotary evaporator. Theblack residue was treated with ether (5 ml) and filtered. The filtratewas dried (Na₂SO₄) and the solvent evaporated with a rotary evaporator,vacuum dried and weighed (300 mg, dark green solid).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 742.

UV-vis (methylene chloride) (λ_(max)(nm)): 764.

IR(KBr): 3435, 3060, 2983, 2932, 2228, 1727, 1603, 1504, 1317, 1256,1218, 1068, 810 cm⁻¹.

EXAMPLE 61 Synthesis of 4,7-diethoxy-1,3-diiminoisoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture of1,4-diethoxy-2,3-phthalonitrile (1.0 g), 25% sodium methoxide inmethanol (1.2 ml), and dry 1-pentanol (20 ml) for 45 minutes. Withcontinued ammonia introduction, the mixture was refluxed for 3 hours.After the resultant had cooled, the solvent was removed with a rotaryevaporator. The residue was dried under vacuum and weighed (1.4 g, greensolid).

EXAMPLE 62 Synthesis of 5,9,14,18,23,27,32,36-octamethoxy2,3-naphthalocyanine (abbreviated as: octamethoxy-2,3-naphthalocyanine)

1,4-dimethoxynaphthalene-2,3-dicarbonitrile (820 mg) suspended in 25%sodium methoxide in methanol (7 ml) was refluxed for 1.5 hours, cooled,and stirred into glacial acetic acid (50 ml). After 30 minutes, thesolvent was evaporated with a rotary evaporator and the residuedissolved in methylene chloride (100 ml). The solution was washedsequentially with 10% hydrochloric acid (100 ml), brine (100 ml) andevaporated with a rotary evaporator. The residue was chromatographed ona silica gel (70-230 mesh, 60 Å, 2×50 cm) column equilibrated intoluene. The product was eluted with toluene, vacuum dried and weighed(52 mg, red-brown solid).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 837.

EXAMPLE 63 Synthesis of Germanium (IV)2,3,9/10,16/17,23/24-tetra-tert-butylphthalocyanine dichloride(abbreviated as: Germanium tetra-tert-butylphthalocyanine dichloride)

Germanium tetrachloride (1.5 ml) was added to a mixture of5-tert-butyl-1,3-diiminoisoindoline (500 mg) and tributylamine (3.4 ml)in 1,2,3,4-tetrahydronaphthalene (7 ml) under an argon atmosphere andthe mixture refluxed for 3.5 hours. The resultant was allowed to cool,treated sequentially with water (20 ml) and methylene chloride (20 ml).The organic layer was separated, washed with water (10 ml), dried(MgSO₄) and evaporated with a rotary evaporator. The residue waschromatographed on a silica gel (70-230 mesh, 60 Å, 2×50 cm) columnequilibrated in toluene. The product was eluted sequentially withtoluene and toluene:isopropanol (9:1), vacuum dried and weighed (310mg).

UV-vis(tetrahydrofuran) (λ_(max)(nm)): 680.

Fluorescence(tetrahydrofuran) (λ_(max)(nm)): 718, 750.

EXAMPLE 64 Effect of Human Serum and Blood on the FluorescenceIntensities of Various Dye Systems in Latex with Different Stokes Shiftsand Excitation and Emission Wavelengths

Donor and acceptor dye pairs or a hybrid phthalocyanine derivative wereincorporated into 0.2 micron latex (CML from IDC, Portland, Oreg.) usingthe tetrahydrofuran solvent system method as indicated in Table 3 and inExample 10. The latex particles were diluted to various solidsconcentrations as indicated in the Table into either a buffer containing5 mM potassium phosphate, 1 mM potassium borate, and 5 mg/ml bovineserum albumin, pH 7, neat human serum or neat human blood. Theexcitation and emission wavelengths and the corresponding Stokes shiftare as indicated in Table 6.

The results show that the fluorescence intensities measured in neathuman serum and blood are greatly affected when the excitationwavelength is in a region where human serum and blood absorb.Conversely, the fluorescence intensities of latex measured in humanserum and blood are not affected when the excitation wavelength is above646 nm. TABLE 3 Latex Dye System Excitation Emission Stokes FluorescenceSolids (Donor/Acceptor) (nm) (nm) Shift Intensity* (%)Trans-4-[4-(Dibutylamino)styryl]-1- 475 680 205 methyl pyridiniumIodide/Silicon phthalocyanine bis(dimethylvinylsilyloxide) Buffer 3690.0019 Serum 28 0.0019 WholeBlood 48 0.0019 Meso-tetra-2-aminophenyl 420680 260 porphine/Silicon phthalocyanine bis(dimethylvinylsilyloxide)Buffer 257 0.0010 Serum 72 0.0010 WholeBlood 11 0.0010(E,E)-3,5-bis-(4-phenyl-1,3- 670 780 110butadienyl)-4,4-difluoro-4-bora- 3a,4a-diazo-s-indacene/Silicon 2,3-naphthalocyanine bis(dimethylhexylvinylsilyloxide) Buffer 21 0.0005Serum 20 0.0005 WholeBlood 22 0.0005 1,1′-Dihexyl-3,3,3′,3′- 650 780 130tetramethylindodicarbocyanine iodide/Silicon 2,3-naphthalocyaninebis(dimethylhexylvinylsilyloxide) Buffer 29 0.0005 Serum 30 0.0005WholeBlood 31 0.0005 Silicon phthalocyanine bis- 670 760 90(dimethylhexylvinylsilyloxide)/Silicon [di(1,6-diphenylnaphthalocyanine)] diphthalocyaninebis(dimethylhexylvinylsilyloxide) Buffer 503 0.0005 Serum 483 0.0005WholeBlood 488 0.0005 Hybrid Compound Silicon [di(1,6- 646 760 114diphenylnaphthalocyanine)] diphthalocyaninebis(dimethylhexylvinylsilyloxide) Buffer 50 0.0007 Serum 45 0.0007WholeBlood 47 0.0007*Fluorescence intensities are not corrected.

EXAMPLE 65 Effect of Axial Ligand on the Quenching ofSilicon[di(1,6-diphenylnaphthalocyanine)]diphthalocyanines

Silicon di(1,6-diphenylnaphthalocyanine)]diphthalocyanine dihydroxideand Silicon[di(1,6-diphenylnaphthalocyanine)]diphthalocyaninebis[dimethylhexylvinylsilyloxide] were incorporated into 0.2 micron CMLlatex (IDC Corporation, Portland Oreg.) at various dye concentrations asindicated in the Table below using the THF solvent system. Thefluorescent latexes were diluted to 0.00057% solids in either 5 mMpotassium phosphate, 1 mm-potassium borate buffer, pH 7 or intetrahydrofuran. The fluorescence intensities were measured byexcitation at 646 nm. Emission was set at 760 nm. The results arepresented below in Table 4.

The results show that the dihydroxy hybrid derivative, which has noaxial ligand, has a large degree of quenching, even at 0.1 mg/ml dyeloading while the bis dimethylhexylvinylsilyloxide hybrid derivative(with the axial ligand) has very little quenching. The results indicatethat axial ligands are important for phthalocyanine derivatives toattain maximum fluorescence intensities in particles. TABLE 4Fluorescence Fluorescence Percent Quench Intensity of Latex PercentQuench of Intensity of Latex of Silicon containing Silicon Silicon[di(1,6- containing Silicon [di(1,6- [di(1,6- diphenylnaphthalocyanine)][di(1,6- Concentration diphenylnaphthalocyanine)]diphenylnaphthalocyanine)] diphthalocyanine diphenylnaphthalocyanine)]of dye per ml of 2% solid diphthalocyanine diphthalocyanine bisdiphthalocyanine bis (mg) dihydroxide dihydroxide[dimethylhexylvinylsilyloxide] [dimethylhexylvinylsilyloxide] 0.1 89 1 04 0.2 75 2 6 7 0.3 80 2 0 10 0.4 78 3 2 13 0.6 82 2 3 16 0.8 84 1 5 19

EXAMPLE 66 Comparison of Quenching in Latex for a Hybrid PhthalocyanineDerivative and a Naphthalocyanine Derivative, Both with Axial Ligands

Silicon[di(1,6-diphenylnaphthalocyanine)]diphthalocyaninebis[dimethylhexylvinylsilyloxide] (hybrid phthalocyanine derivative) andsilicon 2,3-naphthalocyanine bis[dimethylhexylvinylsilyloxide](naphthalocyanine derivative) were incorporated into 0.2 micron CMLlatex (IDC Corporation, Portland Oreg.) at various dye concentrations asindicated in the Table below using the tetrahydrofuran solvent system.The fluorescent latexes were diluted to 0.00057% solids in either 5 mMpotassium phosphate, 1 mM potassium borate buffer, pH 7 or intetrahydrofuran. The fluorescence intensities were measured atexcitation and emission wavelengths as indicated in the Table below.

The results show that the hybrid phthalocyanine derivative is much moreresistant to quenching than the naphthalocyanine derivative. The resultsshow the special properties of the hybrid phthalocyanine derivatives forattaining improved fluorescence intensities in latex. TABLE 5 Silicon2,3- Fluorescence Fluorescence Percent naphthalocyanine Intensity ofPercent Quench Intensity of Quench bis(dimethylhexylvinylsilyloxide)Latex (Ex. 350 nm (Ex. 350 nm Latex (Ex. 650 nm (Ex. 650 nmconcentration (mg/mL) Em. 780 nm) Em. 780 nm) Em. 780 nm) Em. 780 nm)0.1 11 0 1 15 0.3 34 13 3 30 0.5 41 19 4 34 0.7 63 26 6 41 0.9 31 32 346 1.0 31 28 3 42 2.0 33 36 3 47 Silicon [di(1,6-diphenylnaphthalocyanine)] diphthalocyanine Fluorescence FluorescencePercent bis[dimethylhexylvinylsilyloxide) Intensity of Percent QuenchIntensity of Quench concentration Latex (Ex. 350 nm (Ex. 350 nm Latex(Ex. 650 nm (Ex. 650 nm (mg/ml) Em. 760 nm) Em. 760 nm) Em. 760 nm) Em.760 nm) 0.1 11 0 6 0 0.3 31 0 16 0 0.5 56 0 28 0 0.7 60 0 30 0 0.9 78 039 0 1.0 82 0 41 0 2.0 113 0 58 13

EXAMPLE 67

Incorporation and Characterization of Hybrid Phthalocyanine andPhthalocyanine Derivatives into Particles using Tetrahydrofuran andDimethylformamide Solvent Systems

Hybrid phthalocyanine and phthalocyanine derivatives were incorporatedinto carboxyl-modified latex (CML, Interfacial Dynamics Corp. Inc.,Portland, Oreg.) using the procedures indicated below for the dyes andusing dye concentrations as indicated in Table 6. The fluorescenceintensities of the latex solutions were measured at the excitation andemission wavelengths and at the latex concentrations (% solids) asindicated in Table 6 for each of the solvent systems used,

a. 50% Tetrahydrofuran Solvent System

Tetrahydrofuran, THF, (0.19 ml) was added, dropwise over a 5 minuteperiod, to a stirring solution of 0.67 ml of 1.5% solids of latexparticles at room temperature. The latex suspension was stirred at roomtemperature for an additional 30 minutes to swell the latex. The dyesolution (0.47 ml), which consists of two or three dyes, each at anappropriate concentration in tetrahydrofuran, was added dropwise over 5minutes to the stirred latex solution, to give the loading dyeconcentration (in a 1.33 ml volume) as indicated in Table 6. Thelatex-dye solution was stirred at room temperature for 30 minutes in thedark. The latex solution was then transferred to dialysis tubing(Spectra-por, 12-14,000 molecular weight cutoff, Spectrum, Houston,Tex.) and the dye-latex solution was dialyzed against water for 12-15hours at 4° C. The dye-latex solution was removed from dialysis and the% solids of the solution was calculated from the final volume afterdialysis and the starring solids concentration.

b. 70% Tetrahydrofuran Solvent System

Tetrahydrofuran (0.19 ml) was added, dropwise over a 5 minute period, toa stirring solution of 0.4 ml of 2.5% solids of latex particles at roomtemperature. The latex suspension was stirred at room temperature for anadditional 30 minutes to swell the latex. The dye solution (0.74 ml),which consists of two or three dyes, each at an appropriateconcentration in tetrahydrofuran, was added dropwise over 5 minutes tothe stirred latex solution, to give the loading dye concentration (in a1.33 ml volume) as indicated in Table 6. The latex-dye solution wasstirred at room temperature for 30 minutes in the dark. The latexsolution was dialyzed and analyzed according to the procedures outlinedin the preceding 50% tetrahydrofuran solvent system method.

c. 50% Dimethylformamide Solvent System

Dimethylformamide, DMF, (0.19 ml) was added, dropwise over a 5 minuteperiod, to a stirring solution of 0.67 ml of 1.5% solids of latexparticles at room temperature. The latex suspension was stirred at roomtemperature for an additional 30 minutes to swell the latex. The dyesolution (0.47 ml), which consists of two or three dyes, each at anappropriate concentration in dimethylformamide, was added dropwise over5 minutes to the stirred latex solution, to give the loading dyeconcentration (in a 1.33 ml volume) as indicated in Table 6. Thelatex-dye solution was stirred at room temperature for 30 minutes in thedark. The latex solution was then transferred to dialysis tubing(Spectra-por, 12-14,000 molecular weight cutoff, Spectrum, Houston,Tex.) and the dye-latex solution was dialyzed against water for 12-15hours at 4° C. The dye-latex solution was removed from dialysis and the% solids of the solution was calculated from the final volume afterdialysis and the starting solids concentration.

d. 70% Dimethylformamide Solvent System

Dimethylformamide (0.19 ml) was added, dropwise over a 5 minute period,to a stirring solution of 0.4 ml of 2.5% solids of latex particles atroom temperature. The latex suspension was stirred at room temperaturefor an additional 30 minutes to swell the latex. The dye solution (0.74ml), which consists of two or three dyes, each at an appropriateconcentration in dimethylformamide, was added dropwise over 5 minutes tothe stirred latex solution, to give the loading dye concentration (in a1.33 ml volume) as indicated in Table 6. The latex-dye solution wasstirred at room temperature for 30 minutes in the dark. The latexsolution was then dialyzed and analyzed according to the proceduresoutlined in the preceding 50% dimethylformamide solvent system method.TABLE 6 Fluorescence Intensity of Loading particles made in various dyeconc. Molar Emission % Solid loading solvent system Dye Systems mg/mlRatio (excit.) (latex size) 50% THF 70% THF 50% DMF 70% DMF 1. Siliconphthalocyanine 0.066/0.1 1:1 785 nm 0.00057% 21.6 Not 0.4 Not performedbis(dimethylvinylsilyloxide) + Silicon (670 nm) (0.216 μm) performed2,3-naphthalocyanine bis(dimethylhexylvinylsilyloxide) 2. Siliconphthalocyanine 0.08/0.1 1:1 785 nm 0.00057% 37.8 39.1 13.5 12.7bis(dimethylhexylvinylsilyloxide) + Silicon (670 nm) (0.216 μm)2,3-naphthalocyanine bis(dimethylhexylvinylsilyloxide) 3. Siliconphthalocyanine 0.35/0.5 1:1 760 nm 0.00057% 99.5 118.0 22.7 6.6bis(dimethylhexylvinylsilyloxide) (670 nm) (0.216 μm) Silicon[di(1,6-diphenyl-2,3- naphthalcyanine)] diphthalocyaninebis(dimethylhexylvinylsilyloxide) 4. Silicon phthalocyanine 0.35/0.5/1:1:0.023 785 nm 0.00057% 86.9 105.9 18.5 7.7bis(dimethylhexylvinylsilyloxide) + Silicon 0.1 (670 nm) (0.216 μm)[di(1,6-diphenyl-2,3- naphthalocyanine)] diphthalocyaninebis(dimethylhexylvinylsilyloxide) + Silicon 2,3-naphthalocyaninebis(dimethylhexylvinylsilyloxide)

EXAMPLE 68 Synthesis of 4,7-diphenyl-1,3-diiminoisoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture of3,6-diphenylphthalonitrile (5.9 g), [synthesized according to J. Am.Chem. Soc. 75, 4338 (1953) and J. Org. Chem., USSR (English Translation)8, 341 (1972)], 25% sodium methoxide in methanol (1.35 ml), and dry1-butanol (20 ml) for 1 hour. With continued ammonia introduction, themixture was refluxed for 1.5 hours. After the resultant had cooled theproduct was collected by filtration, washed sequentially with 1-butanol(10 ml) and ether (10 ml), vacuum dried and weighed (0.62 g).

EXAMPLE 69 Synthesis of[2,12-di-(2,3)-naphtho[b,l]-7¹,7⁴,17¹,17⁴-tetraphenyldibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]silicondihydroxide (abbreviated as:Silicon(di(2,3-naphthalocyanine)]di(1,4-diphenylphthalocyanine)dihydroxide))

Silicon tetrachloride (69 μL) was added to a mixture of4,7-diphenyl-1,3-diiminoisoindoline (119 mg) and1,3-diiminobenz[f]isoindoline (39 mg) in freshly distilled quinoline (1ml) under an argon atmosphere and the mixture heated with stirring at200° C. for 1 hour. The resultant was allowed to cool to 160° C.,treated with water (1 ml) and refluxed for 5 minutes. The mixture wascooled, treated with ether (10 ml) and filtered, washing the solidsequentially with water (5 ml) and ether (5 ml). The organic layer ofthe filtrate was separated from the aqueous layer washed sequentiallywith 1 N hydrochloric acid (10 ml) and water (10 ml), dried (MgSO₄) andevaporated with a rotary evaporator. The residue was chromatographed ona silica gel (70-230 mesh, 60 Å, 2×50 cm) column equilibrated inmethylene chloride. The product was eluted with methylene chloride—1%isopropanol, vacuum dried and weighed (43 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 690, 736, 758

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 774

EXAMPLE 70 Synthesis of[2,12-di-(2,3)-naphtho[b,l]-7¹,7⁴,17¹,17⁴-tetraphenyldibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(7-oct-1-enyldimethylsilyloxide), (abbreviated as: Silicon(di(2,3-naphthalocyanine)]di(1,4-diphenylphthalocyanine)bis(dimethylhexylvinylsilyloxide))

A mixture ofsilicon[di(2,3-naphthalocyanine)]di(1,4-diphenylphthalocyanine)dihydroxide (10.6 mg), 7-oct-1-enyldimethylchlorosilane (41 μL),imidazole (11 mg) and dimethylformamide (200 μL) was stirred at roomtemperature for 30 minutes. The resultant was concentrated under vacuumon the rotary evaporator. The residue was chromatographed on a silicagel (70-230 mesh, 60 Å, 2×50 cm) column equilibrated in hexane. Theproduct was eluted with toluene, vacuum dried and weighed (3 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 667, 745

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 774

EXAMPLE 71 Synthesis of[2,12-di-(2,3)-naphtho[b,l]-7¹,7⁴,17¹,17⁴-tetraphenyldibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(dimethylpentafluorophenylsilyloxide), (abbreviated as:Silicon[di(2,3-naphthalocyanine]di(1,4-diphenylphthalocyanine)bis(dimethylpentafluorophenylsilyloxide))

A mixture ofsilicon[di(2,3-naphthalocyanine)]di(1,4-diphenylphthalocyanine)dihydroxide (10 mg), chlorodimethylpentafluorophenylsilane (28 μL),imidazole (10 mg) and dimethylformamide (200 μL) was stirred at roomtemperature for 10 minutes. The resultant was concentrated under vacuumon the rotary evaporator. The residue was chromatographed on a silicagel (70-230 mesh, 60 Å, 2×50 cm) column equilibrated in hexane. Theproduct was eluted with toluene, vacuum dried and weighed (3 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 701, 754

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 789

EXAMPLE 72 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7^(2/3),17^(2/3)-di(tert-butyl)dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]silicondihydroxide (abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)]di2,3-tert-butylphthalocyanine) dihydroxide))

Silicon tetrachloride (344 μL) was added to a mixture ofdiphenyl-1,3-diiminobenz[f]isoindoline (869 mg) and5-tert-butyl-1,3-diiminoisoindoline (100.5 mg) in freshly distilledquinoline (2 ml) under an argon atmosphere and the mixture heated withstirring at 200° C. for 1 hour. The resultant was allowed to cool to150° C., treated with water (3 ml) and refluxed for 10 minutes. Themixture was cooled, treated with ether (30 ml) and filtered, washing thesolid sequentially with ether (20 ml) and water (20 ml). The organiclayer of the filtrate was separated from the aqueous layer, washedsequentially with 1N hydrochloric acid (2×10 ml) and water (10 ml),dried (MgSO₄) and evaporated with a rotary evaporator. The residue waschromatographed three times on a silica gel (70-230 mesh, 60 Å, 2×50 cm)column equilibrated in hexane. The product was eluted sequentially withmethylene chloride and methylene chloride—1% isopropanol, vacuum driedand weighed (55 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 646, 684, 720, 743

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 750

EXAMPLE 73 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7^(2/3),17^(2/3)-di(tert-butyl)dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(7-oct-1-enyldimethylsilyloxide) (abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)]di(2,3-butylphthalocyaninebis(dimethylhexylvinylsilyloxide))

A mixture of Silicondi(1,6-diphenyl-2,3-naphthalocyanine)]di(2,3-tert-butylphthalocyanine)dihydroxide(2.8 mg) and dimethylformamide (500 μL) was stirred at room temperaturefor 10 minutes. The resultant was concentrated under vacuum on therotary evaporator. The residue was chromatographed on a silica gel(70-230 mesh, 60 Å, 2×50 cm) column equilibrated in hexane. The productwas eluted sequentially with hexane and toluene, vacuum dried andweighed (16.5 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 648, 688, 726, 750

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 756

EXAMPLE 74 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7^(2/3),17^(2/3)-di(tert-butyl)dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(dimethylpentafluorophenylsilyloxide) (abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)]di(2,3-tert-butylphthalocyanine)bis(dimethylpentafluorophenylsilyloxide))

A mixture ofSilicon[di(1,6-diphenyl-2,3-naphthalocyanine)]di(2,3-tert-butylphthalocyanine)dihydroxide(21.8 mg), chlorodimethylpentafluorophenylsilane (56.5 μL), imidazole(20.4 mg) and dimethylformamide (500 μL) was stirred at room temperaturefor 10 minutes. The resultant was concentrated under vacuum on therotary evaporator. The residue was chromatographed on a silica gel(70-230 mesh, 60 Å) column (2×50 cm) equilibrated in hexane. The productwas eluted sequentially with hexane and toluene, vacuum dried andweighed (25 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 652, 694, 730, 760

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 769

EXAMPLE 75 Synthesis of[2¹,2⁶,12¹,12⁶,-tetraphenyldinaphtho[b,l]-7-(2,3)-naphtho[g]-17-benzo[q]-5,10,15,20-tetraazoporphyrinato]silicondihydroxide (abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)](2,3-naphthalocyanine)phthalocyaninedihydroxide)

Silicon tetrachloride (172 μL) was added to a mixture ofdiphenyl-1,3-diiminobenz[f]isoindoline (347 mg),1,3-diiminobenz[f]isoindoline (49 mg) and 1,3-diiminoisoindoline (36 mg)in freshly distilled quinoline (2 ml) under an argon atmosphere and themixture heated with stirring at 200° C. for 1 hour. The resultant wasallowed to cool to 170° C., treated with water (2 ml) and refluxed for 5minutes. The mixture was cooled, treated with ether (20 ml) andfiltered, washing the solid sequentially with water (5 ml) and ether (10ml). The organic layer was separated from the aqueous layer, washed with1 N hydrochloric acid (2×10 ml), (filtering again to effect separation)and water (10 ml), dried (MgSO₄) and evaporated with a rotaryevaporator. The residue was chromatographed on a silica gel (70-230mesh, 60 Å) column (2×50 cm) equilibrated in hexane. The product waseluted sequentially with toluene, toluene—5% methylene chloride,toluene—10% methylene chloride, toluene—20% methylene chloride andfinally toluene—50% methylene chloride. The product was thenrechromatographed on silica gel (GF, 1000μ, 20×20 cm) plates elutingsequentially (air drying the plates between each elution) withtoluene—5% methylene chloride, toluene—10% methylene chloride,toluene—20% methylene chloride and finally toluene—50% methylenechloride. The plates were eluted in the latter solvent ten times toeffect separation of the desired product from by-products. The greenproduct was vacuum dried and weighed (9 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 670, 714, 750

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 762

EXAMPLE 76 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7-(2,3)-naphtho[g-17-benzo[q]-10,15,20,-tetraazoporphyrinato]siliconbis(7-oct-1-enyldimethylsilyloxide) (abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)](2,3-naphthalocyanine)phthalocyaninebis(dimethythexylvinylsilyloxide))

A mixture of[di(1,6-diphenyl-2,3-naphthalocyanine)](2,3-naphthalocyanine)phthalocyaninedihydroxide (9 mg), 7-oct-1-enyldimethylchlorosilane (33.5 μL),imidazole (9 mg) and dimethylformamide (200 μL) was stirred at roomtemperature for 10 minutes. The resultant was concentrated under vacuumon the rotary evaporator. The residue was chromatographed on asilica-gel (GF, 1000μ, 20×20 cm) plate eluting with hexane—50% methylenechloride. The product was triturated twice with hexane (1 ml), vacuumdried and weighed (9 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 674, 718, 756

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 763

EXAMPLE 77 Synthesis of[2¹,2⁶-diphenylnaphtho[b]-7,12,17-tribenzo[g,l,q]-5,10,15,20-tetraazoporphyrinato]silicondihydroxide (abbreviated as: Silicon(1,6-diphenyl-2,3-naphthalocyanine)triphthalocyanine dihydroxide)

Silicon tetrachloride (687 μL) was added to a mixture ofdiphenyl-1,3-diiminobenz[f]isoindoline (347 mg) and1,3-diiminoisoindoline (726 mg) in freshly distilled quinoline (5 ml)under an argon atmosphere and the mixture heated with stirring at 200°C. for 1 hour. The resultant was allowed to cool to 170° C., treatedwith water (5 ml) and refluxed for 5 minutes. The mixture was cooled,treated with ether (20 ml) and filtered, washing the solid sequentiallywith water (10 ml) and ether (10 ml). The organic layer was separatedfrom the aqueous layer, washed sequentially with 1N hydrochloric acid(50 ml), (re-filtering to effect separation) and water (50 ml), dried(MgSO₄) and evaporated with a rotary evaporator. The filtered solidswere treated with acetone (20 ml) and re-filtered washing with acetone(10 ml). The filtrate was dried (MgSO₄) and evaporated with a rotaryevaporator. The residues from the ether and acetone evaporations werecombined and chromatographed on a silica gel (70-230 mesh, 60 Å) column(2×50 cm) equilibrated in hexane. The product was eluted sequentiallywith methylene chloride, toluene and toluene-1% isopropanol. The productwas then rechromatographed on silica gel (GF, 1000μ, 20×20 cm) plateseluting with methylene chloride, air drying the plates and re-elutingwith toluene—1% isopropanol. The blue-green product was vacuum dried andweighed (60 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 622, 658, 688, 698

EXAMPLE 78 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(trihexylsilyloxide) (abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)]diphthalocyaninebis(trihexylsilyloxide))

A mixture of Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)]dihydroxide(8 mg), chlorotrihexylsilane (55 μL), imidazole (10 mg) anddimethylformamide (200 μL) was stirred at room temperature for 10minutes. The resultant concentrated under vacuum on the rotaryevaporator. The residue was chromatographed on a silica gel (70-230mesh, 60 Å) column (2×50 cm) equilibrated in hexane. The product waseluted sequentially with hexane and toluene, vacuum dried and weighed(4.5 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 6.44, 684, 718, 748

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 752

EXAMPLE 79 Synthesis of[2¹,2⁶-diphenylnaphtho[b]-7,12,17-tribenzo[g,l,q]5,10,15,20-tetraazoporphyrinato]siliconbis(7-oct-1-enyldimethylsilyloxide) (abbreviated as:Silicon(1,6-diphenyl-2,3-naphthalocyanine triphthalocyaninebis(dimethylhexylvinylsilyloxide))

A mixture of Silicon(1,6-diphenyl-2,3-naphthalocyanine)triphthalocyanine dihydroxide (23.3mg), 7-oct-1-enyldimethylchlorosilane (115.2 μL), imidazole (30.6 mg)and dimethylformamide (500 uL) was stirred at room temperature for 10minutes. The resultant was concentrated under vacuum on the rotaryevaporator. The residue was treated with hexane (2 ml), filtered fromyellow insoluble solid and the filtrate evaporated. The residue waschromatographed on a silica gel (GF, 1000μ, 20×20 cm) plate eluting withhexane, air drying the plate and re-eluting with hexane—50% methylenechloride. The product was vacuum dried and weighed (0.8 mg).

NMR(500 MHZ, CDCl₃) δ9.54(m,2H), 9.47(d,2H), 8.41 (d,2H), 8.37(m,2H),8.25(m,2H), 8.19(dd,2H), 8.09(dd,2H), 8.02(m,10H), 5.65(m,2H),4.90(m,4H), 1.67(m,4H), 0.76(m,4H), −0.11(m,4H), −1.25 (m,4H), −2.17(m,4H), −2.79(s,12H).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 624, 660, 692

Fluorescence (tetrahydrofuran) (λ_(max)(nm): 710

EXAMPLE 80 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconhis (7-oct-1-enyldimethylsilyloxide) (abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)]di(2,3-naphthalocyanine)bis(dimethylhexylvinylsilyloxide))

A mixture ofSilicon[di(1,6-diphenyl-2,3-naphthalocyanine)]di(2,3-naphthalocyanine)dihydroxide(6 mg), 7-oct-1-enyldimethylchlorosilane (21 μL), imidazole (5.7 mg) anddimethylformamide (200 μL) was stirred at room temperature for 10minutes. The resultant was concentrated under vacuum on the rotaryevaporator. The residue was chromatographed on a silica gel (GF, 1000μ,20×20 cm) plate eluting sequentially (air drying the plate between eachelution) with hexane—20% toluene, hexane—50% toluene and toluene. Thegreen product was triturated three times with hexane (1 ml), vacuumdried and weighed (5.4 mg).

NMR (500 MHZ, CDCl₃) δ8.75 (b,4H), 8.38(m,8H), 8.15(m,4H), 8.03(m,16H),7.80(m,8H), 5.40(m,2H), 4.70(m,4H), 1.38(m,4H), 0.59(m,4H), 0.16(m,4H),−0.05(m,4H), −1.08(m,4H), −1.97(m,4H), −2.58(s,1 2H).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 668, 696, 746, 784

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 792

EXAMPLE 81 5,6-dicyano-1,3-diiminoisoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture ofbenzene-1,2,4,5-tetracarbonitrile (1.78 g), and dry methanol (40 ml) for1 hour. The product was collected by filtration, washed sequentiallywith methanol (10 ml) and ether (10 ml), vacuum dried and weighed (2.07g).

EXAMPLE 82 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7²,7³17²,17³-tetracyanodibenzo[[g,q]-5,10,15,20-tetraazoporphyrinato]silicondihydroxide

Silicon tetrachloride (115 μL) was added to a mixture ofdiphenyl-1,3-diiminobenz[f]isoindoline (174 mg) and5,6-dicyano-1,3-diiminoisoindoline (98 mg) in freshly distilledquinoline (2 ml) under an argon atmosphere and the mixture heated withstirring 200° C. for 1 hour. The resultant was allowed to cool to 170°C. treated with water (2 ml) and refluxed for 5 minutes. The mixture wascooled, treated with ether (20 ml) and filtered, washing the solidsequentially with water (10 ml) and ether (10 ml). The filtered darkgreen insoluble solid was treated with acetone (20 ml), filtered,treated with methylene chloride (20 ml) and re-filtered washing withmethylene chloride (20 ml). The acetone/methylene chloride filtrate wasdried (MgSO₄) and evaporated with a rotary evaporator. The residue waschromatographed on a silica gel (70-230 mesh, 60 Å) column (2×50 cm)equilibrated in hexane. The product was eluted sequentially withmethylene chloride and methylene chloride—1% isopropanol, vacuum driedand weighed (63 mg).

IR (KBr) 2233 cm⁻¹ (CN)

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 627, 686, 746, 826

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 831

EXAMPLE 83 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7²,7³,17²,17³-tetraacyanodibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(7-oct-1-enyldimethylsilyloxide)(abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)]di-(2,3-dicyanophthalocyanine)bis(dimethylhexylvinylsilyloxide))

A mixture ofSilicon[di(1,6-diphenyl-2,3-naphthalocyanine)]di(2,3-dicyanophthalocyanine)dihydroxide(21.6 mg), 7-oct-1-enyldimethylchlorosilane (77 μL), imidazole (20.4 mg)and dimethylformamide (500 μL) was stirred at room temperature for 10minutes. The resultant was concentrated under vacuum on the rotaryevaporator. The residue was chromatographed on a silica gel (GF, 1000μ,20×20 cm) plate eluting with hexane, air drying the plate and re-elutingwith methylene chloride. The product was vacuum dried and weighed (4mg).

NMR (500 MHZ, CDCl₃) δ8.65 (s,4H), 8.38(m,4H), 8.16(m,4H), 8.02(m,4H),7.94(m,8H), 7.87(m,4H), 5.51(m,2H), 4.81(m,4H), 1.55(m,4H), 0.71(m,4H),0.24(m.4H), −0.06(m,4H), −1, 19(m,4H), −2.07(m,4H), −2.71 (s,2H)

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 631, 693, 752, 835

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 839

EXAMPLE 84 Synthesis of[2,7/12-di-(2,3)-naphtho[b,g/l)-7²,7³,12²,12/17²,17³tetracyanodibenzo[g,l/q]-5,10,15,20-tetraazoporphyrinato]silicondihydroxide (abbreviated as: Silicon[di(2,3-naphthalocyanine)]di(2,3-dicyanophthalocyanine)dihydroxide)

Silicon tetrachloride (330 μL) was added to a mixture of1,3-diiminobenz[f]isoindoline (195 mg) and5,6-dicyano-1,3-diiminoisoindoline (195 mg) in freshly distilledquinoline (4 ml) under an argon atmosphere and the mixture heated withstirring at 200° C. for 1 hour. The resultant was allowed to cool to160° C., treated with water (4 ml) and refluxed for 10 minutes. Themixture was cooled, treated with ether (20 ml) and filtered, washing thesolid sequentially with water (10 ml), ether (10 ml), and acetone (10ml). The solid was vacuum dried and weighed (560 mg).

EXAMPLE 85 Synthesis of[2,7/12-di-(2,3)-naphtho[b,g/l]-7²,7³,12²,12³/17²,17tetracyanodibenzo[g,l/q]-5,10,15,20-tetraazoporphyrinato]siliconbis(7-oct-1-enyldimethylsilyloxide) (abbreviated as:Silicon[di(2,3-naphthalocyanine)]di(2,3-dicyanophthalocyanine)bis(dimethylhexylvinylsilyloxide))

A mixture ofSilicon[di(2,3-naphthalocyanine)]di(2,3-dicyanophthalocyanine)dihydroxide (155 mg), 7-oct-1-enyldimethylchlorosilane (770 ml),imidazole (204 mg) and dimethylformamide (2 ml) was stirred at roomtemperature for 30 minutes. The resultant was concentrated under vacuumon the rotary evaporator. The residue was chromatographed on two silicagel (GF, 2000μ, 20×20 cm) plates eluting with hexane, air drying theplate and re-eluting with methylene chloride. The product was vacuumdried and weighed (3.1 mg).

NMR (500 MHZ, CDCl³) δ10.3(s,4H), 9.94(s,4H), 8.65(m,4H), 7.98(m,4H),5.80(m,1H), 5.59(m,1H), 4.92 (m,4H), 1.56(m,4H), 0.71(m,4H), 0.26(m,4H),−0.05(m,4H), −0.96(m,4H), −1.83(m,4H), −2.44, (s, 12H)

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 649, 704, 731, 788

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 795

EXAMPLE 86 Synthesis of[2,7/12-di-(2,3)-naphtho[b,g/l]-7²,7³,12²,12³/17²,17tetracyanodibenzo[g,l/q]-5,10,15,20-tetraazoporphyrinato]siliconbis(dimethylpentafluorophenylsilyloxide) (abbreviated as:Silicon[di(2,3-naphthalocyanine)]di(2,3-dicyanophthalocyanine)bis(dimethylpentafluorophenylsilyloxide))

A mixture ofSilicon[di(2,3-naphthalocyanine)][di(2,3-dicyanophthalocyanine)]dihydroxide(155 mg), chlorodimethylpentafluorophenylsilane (565 μL), imidazole (204mg) and dimethylformamide (2 ml) was stirred at room temperature for 1hour. The resultant was concentrated under vacuum on the rotaryevaporator. The residue was chromatographed on two silica gel (GF, 1000μ20×20 cm) plates eluting with hexane, air drying the plate andre-eluting with methylene chloride, vacuum dried and weighed (3 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 656, 712, 740, 800

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 807

EXAMPLE 87 Synthesis of 5,6-dichloro-1,3-diiminoisoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture of4,5-dichlorphthalonitrile (1.0 g), 8% sodium butoxide in 1-butanol (500μL), 1,4-dioxane (1 ml), and dry 1-butanol (10 ml) for 60 minutes. Withcontinued ammonia introduction, the mixture was refluxed for 2 hours.After the resultant had cooled, the product was collected by filtration,washed with methylene chloride (20 ml), vacuum dried and weighed (0.63g).

EXAMPLE 88 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7²,7³,17²,17³-tetrachlorodibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]silicondihydroxide (abbreviated as:Silicon[di-(1,6-diphenyl-2,3-naphthalocyanine)]di(2,3-dichlorophthalocyanine)dihydroxide)

Silicon tetrachloride (500 μL) was added to a mixture of5,6-dichloro-1,3-diiminoisoindoline (308 mg) and4,7-diphenyl-1,3-diiminobenz[f]isoindoline (900 mg) in freshly distilledquinoline (14 ml) under an argon atmosphere and the mixture heated withstirring at 210° C. for 1 hour. The resultant was allowed to cool to160° C., treated with water (3 ml) and refluxed for 10 minutes. Themixture was cooled, treated with ether (50 ml) and filtered, washing thesolid sequentially with water (50 ml) and ether (100 ml). The organiclayer of the filtrate was separated from the aqueous layer, washedsequentially with 1 N hydrochloric acid (50 ml) and water (100 ml) andevaporated with a rotary evaporator. The residue was chromatographed ona silica gel (70-230 mesh, 60 Å) column (2×50 cm) equilibrated inhexane. The product was eluted with toluene—10% isopropanol, vacuumdried and weighed (340 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 716, 766, 694.

EXAMPLE 89 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7²,7³,17²,17³-tetrachlorodibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(7-oct-1-enyldimethylsilyloxide), (abbreviated as:Silicon[di(1,6-diphenyl-2,3-naphthalocyanine)]di(2,3-dichlorophthalocyanine)bis(dimethylhexylvinylsilyloxide))

A mixture ofsilicon[di(1,6-diphenyl-2,3-naphthalocyanine)]di(2,3-dichlorophthalocyanine)dihydroxide(340 mg), 7-oct-1-enyldimethylchlorosilane (1.1 ml), imidazole (325 mg)and dimethylformamide (7 ml) was stirred at room temperature for 48hours. The resultant was concentrated under vacuum on the rotaryevaporator. The residue was chromatographed on a silica gel (70-230mesh, 60 Å) column (2×50 cm) equilibrated in hexane. The product waseluted with toluene, vacuum dried and weighed (75 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 720, 770, 698.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 781

EXAMPLE 90 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo(g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(octyloxide) (abbreviated as: Silicondi[(1,6-diphenyl)-2,3-naphthalocyanine)]diphthalocyanine bis(octyloxide)

A mixture of Silicondi(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyanine dihydroxide (49mg) and 1-octanol (1 ml) was refluxed with stirring on an oil bath at235° C. for 3 hours. The resultant was concentrated under vacuum on therotary evaporator (using a water bath at 60° C.). The residue waschromatographed on two silica gel (GF, 1000μ 20×20 cm) plates elutingwith methylene chloride three times (air drying the plates between eachelution). The product was vacuum dried and weighed (19 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 642, 682, 716, 746

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 751

EXAMPLE 91 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo(g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(octyloxide) (abbreviated as: Silicondi[(1,6-diphenyl)-2,3-naphthalocyanine)]diphthalocyanine bis(phenoxide)

A mixture of Silicondi[(1,6-diphenyl)-2,3-naphthalocyanine}diphthalocyanine dihydroxide (49mg), and phenol (1 g) was refluxed with stirring on an oil bath at 220°C. for 2 hours. The resultant was allowed to cool and chromatographed ona silica gel (70-230 mesh, 60 Å) column (2×50 cm) equilibrated inhexane. The product was eluted with hexane—50% methylene chloride,vacuum dried and weighed (13 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 654, 704, 732, 768

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 776

EXAMPLE 92 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo(g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis[polyethylene glycol)methyl ether] (abbreviated as: Silicondi(1,6-diphenyl-2,3 naphthalocyanine))diphthalocyanine bis[poly(ethyleneglycol)methyl ether])

A mixture ofSilicon[di(1,6-diphenyl-2,3-naphthalocyanine)]diphthalocyaninedihydroxide (49 mg), poly(ethyleneglycol)methyl ether (400 mg), and1,2,4-trimethylbenzene (5 ml) was refluxed with stirring on an oil bathat 220° C. for 3 days using a Dean-Stark trap. The resultant wasconcentrated under vacuum on the rotary evaporator. The residue waschromatographed on a silica gel (70-230 mesh, 60 Å) column (2×50 cm)equilibrated in methylene chloride and eluted sequentially withmethylene chloride—1% isopropanol, methylene chloride—5% isopropanol,methylene chloride—20% isopropanol, methylene chloride—50% isopropanoland finally methylene chloride—50% methanol. The product was vacuumdried and weighed (145 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 648, 692, 726, 758

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 765

EXAMPLE 93 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo(g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis[(4-octyl)phenoxide](abbreviated as: Silicon[di(1,6-diphenyl-2,3naphthalocyanine)]diphthalocyanine bis[(4-octyl)phenoxide])

A mixture ofSilicon[di(1,6-diphenyl-2,3-naphthalocyanine)]diphthalocyaninedihydroxide (42 mg), 4-octylphenol (41 mg) and 1,2,4-trimethylbenzene (5ml) was refluxed with stirring on an oil bath at 200° C. for 16 hours.The resultant was concentrated under vacuum on the rotary evaporator.The residue was chromatographed on a silica gel (70-230 mesh, 60 Å)column (2×50 cm) equilibrated in hexane and eluted with hexane—50%methylene chloride. The product was vacuum dried and weighed (49 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 644, 684, 716, 746

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 751

EXAMPLE 94 Silicon 2,3-naphthalocyanine bis(dimethyloctadecylsilyloxide)

A mixture of Silicon 2,3-naphthalocyanine dihydroxide (155 mg),chlorodimethyloctadecylsilane (1.04 g), imidazole (204 mg) anddimethylformamide (5 μL) was stirred at room temperature for 1 hour. Theresultant was concentrated under vacuum on the rotary evaporator. Theresidue was chromatographed on a silica gel (70-230 mesh, 60 Å) column(2×50 cm) equilibrated in hexane. The product was eluted sequentiallywith hexane and methylene chloride, vacuum dried and weighed (180 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 686, 732, 770

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 776

EXAMPLE 95 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo(g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis[poly(ethylene glycol)(abbreviated as: Silicondi[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyaninebis[poly(ethylene glycol)])

A mixture of Silicon di[(1,6-diphenyl)-2,3naphthalocyanine]diphthalocyanine dihydroxide (49 mg), poly(ethyleneglycol) (1 g), and 1,2,4-trimethylbenzene (5 ml) was refluxed withstirring on an oil bath at 210° C. for 3 days using a Dean-Stark trap.The resultant was concentrated under vacuum on the rotary evaporator.The residue was chromatographed on a silica gel (70-230 mesh, 60 Å)column (2×50 cm) equilibrated in methylene chloride and elutedsequentially with methylene chloride—1% isopropanol, methylenechloride—5% isopropanol, methylene chloride—20% isopropanol and finallymethylene chloride—50% isopropanol. The product was thenre-chromatographed on silica gel GF, 1000μ, 20×20 cm) plates elutingsequentially (air drying the plates between each elution) with methylenechloride, methylene chloride-10% methanol and finally tetrahydrofuran.The product was vacuum dried and weighed (152 mg). NMR (500 MHZ, CDCl₃)δ8.30(m,4H), 8.25 (m,4H), 8.00(m,24H,) 7.77(m,4H), 3.63(m,CH₂'s).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 648, 692, 720, 754

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 760

EXAMPLE 96 Synthesis of[2¹2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo(g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis[poly(ethylene glycol)] (abbreviated as: Silicondi(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyanine[poly(ethyleneglycol)][poly(ethylene glycol)acetylthiopropionate])

A mixture of acetylthiopropionic acid, (15 mg), 1,1′-carbonyldiimidazole(16 mg) and dimethylformamide (1 ml) was stirred at room temperature for40 minutes. A portion of this solution (100 μL) was added to Silicondi[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyaninebis[poly(ethylene glycol)](49.5 mg) and the mixture stirred at roomtemperature for 3 days. The resultant was concentrated under vacuum onthe rotary evaporator. The residue was chromatographed on a silica gel(GF, 1000μ, 20×20 cm) plate eluting with tetrahydrofuran, vacuum driedand weighed (3 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 644, 690, 718, 750

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 754

EXAMPLE 97 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7²,7³,17²,17³-tetracarboxydibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]silicondihydroxide (abbreviated as: Silicondi[(1,6-diphenyl)2,3-naphthalocyanine]di(2,3-dicarboxyphthalocyanine)dihydroxide)

A mixture of Silicondi[(1,6-diphenyl)-2,3-naphthalocyanine]di(2,3-dicyanophthalocyanine)dihydroxide(36 mg) and concentrated sulfuric acid (200 μL) was heated with stirringat 50° C. for 48 hours. The cooled mixture was then carefully treatedwith water (150 μL) and heated with stirring at 100° C. for 20 hours.The cooled mixture was then treated with water (1 ml) and the darkprecipitate collected by filtration washing with water (1 ml). The solidwas then treated with 1 N potassium carbonate solution (1 ml) andrefluxed with stirring for 1 hour. The cooled mixture was acidified topH 2 by dropwise addition of 6 N hydrochloric acid and the fine darkgreen solid product filtered, washing with water (1 ml). The solid wasvacuum dried and weighed (20 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 636, 658, 716, 788

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 791

EXAMPLE 98 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7²,7³,17²,17³-tetracarboxydibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis[poly(ethylene glycol)methyl ether](abbreviated as: Silicondi[(1,6-diphenyl)2,3-naphthalocyanine]di(2,3-dicarboxyphthalocyanine)bis[poly(ethyleneglycol)methyl ether])

A mixture of Silicondi[(1,6-diphenyl)2,3-naphthalocyanine]di(2,3-dicarboxyphthalocyanine)dihydroxide(10 mg), poly(ethylene glycol)methyl ether]) (80 mg) and1,2,4-trimethylbenzene (1 ml) was refluxed with stirring on an oil bathat 220° C. for 3 days using a Dean-Stark trap. The resultant wasconcentrated under vacuum on the rotary evaporator. The residue waschromatographed on a silica gel (GF, 1000μ 20×20 cm) plate eluting withmethylene chloride—10% methanol, air drying the plate and re-elutingwith methylene chloride—10% methanol. The green product was vacuum driedand weighed (8 mg).

IR (KBr)1712 cm⁻¹ (COOH)

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 648, 702, 726, 792.

UV-vis (Water) (λ_(max)(nm)): 712, 816.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 800.

EXAMPLE 99 Synthesis of Silicon (IV) 2,3-naphthalocyaninebis(tert-butyldimethylsilyloxide)

A mixture of silicon naphthalocyanine dihydroxide,tertbutyldimethylchlorosilane (390 mg), imidazole (180 mg) anddimethylformamide (5 ml) was stirred at 150° C. for 30 minutes. Theresultant was chromatographed on a silica gel (70-230 mesh, 60 Å) column(2×50 cm) equilibrated in hexane. The product was eluted sequentiallywith hexane and toluene, vacuum dried and weighed (6 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 772, 730, 686.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 775.

¹H-NMR (500 MHZ, CDCl₃,) δ 10.14 (s,8H), 8.67(m,8H), 7.90(m,8H),−1.20(s,18H), −2.60(s,12H)

EXAMPLE 100 Synthesis of Silicon (IV) phthalocyaninebis(tert-butyl-dimethylsilyloxide)

A mixture of Silicon (IV) phthalocyanine dihydroxide (200 mg),tert-butyldimethylchlorosilane (525 mg), imidazole (272 mg) anddimethylformamide (5 ml) was stirred at 150° C. for 30 minutes. Theresultant was chromatographed on a silica gel (70-230 mesh, 60 Å) column(2×50 cm) equilibrated in hexane. The product was eluted sequentiallywith hexane and toluene, vacuum dried and weighed (12 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 666, 636, 600.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 671.

¹H-NMR (500 MHZ, CDCl₃,) 69.65(m,8H), 8.33(m,8H), −1.45(s,18H),−2.98(s,12H)

EXAMPLE 101 Synthesis of[2¹2²-dichlorobenzo[b]-7,12,17-tri(2,3-naphtho)[g,l,q]-5,10,15,20,-tetraazoporphyrinato]silicondihydroxide (abbreviated as:Silicon[tri(2,3-naphthalocyanine)]2,3-dichlorophthalocyaninedihydroxide)

Silicon tetrachloride (600 μL) was added to a mixture of 5,6-dichloro1,3-diiminoisoindoline (100 mg) and 1,3-diiminobenz[f]isoindoline (466mg) in freshly distilled quinoline (4 ml) under an argon atmosphere andthe mixture heated with stirring at 210° C. for 2 hours. The resultantwas allowed to cool, treated with water (20 ml) and refluxed for 20minutes. The mixture was cooled, treated with ether (10 ml) andfiltered, the solid was washed sequentially with water (2×20 ml), ether(3×20 ml), methylene chloride (10 ml) and acetone (20 ml). The solid wasvacuum dried and weighed (0.83 g). The crude product was used withoutpurification for the next step.

EXAMPLE 102 Synthesis of[2¹2²-dichlorobenzo[b]-7,12,17-tri(2,3-naphtho)[g,l,q]-5,10,15,20,-tetraazoponphyrinato]siliconbis(7-oct-1-enyldimethylsilyloxide) (abbreviated as:Silicon[tri(2,3-naphthalocyanine)]2,3-dichlorophthalocyaninebis(dimethylhexylvinylsilyloxide))

A mixture of silicon[tri(2,3-naphthalocyanine)2,3-dichlorophthalocyaninedihydroxide (400 mg) and 7-oct-1-enyldimethylchlorosilane (1.5 ml) wasstirred at room temperature for 15 hours. The resultant was concentratedunder vacuum on the rotary evaporator. The residue was chromatographedon a silica gel (70-230 mesh, 60 Å) column (2×50 cm) equilibrated inhexane. The product was eluted with toluene, vacuum dried and weighed(35 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm) ε (M⁻¹cm⁻¹)): 770, 728, 688, 654,182000.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 774, 727.

EXAMPLE 103 Synthesis of[2¹,2²-dichlorobenzo[b]-7,12,17-tri(2,3-naphtho)[g,l,q]-5,10,15,20,-tetraazoporphyrinato]siliconbis(dimethylpentafluorophenylsilyloxide)] (abbreviated as:Silicon[tri(2,3-naphthalocyanine)]2,3-dichlorophthalocyaninebis(dimethylpentafluorophenylsilyloxide))

A mixture ofsilicon[tri(2,3-naphthalocyanine)]2,3-dichlorophthalocyanine dihydroxide(400 mg), chlorodimethylpentafluorophenylsilane (1.0 ml), imidazole (270mg) and dimethylformamide (5 ml) was stirred at room temperature for 16hours. The reaction mixture was filtered, washing the solid withdimethylformamide (4×2 ml). The filtrate was evaporated under vacuum onthe rotary evaporator. The residue was dissolved in toluene andfiltered. The filtrate was concentrated under vacuum on the rotaryevaporator. The residue was chromatographed on a silica gel (70-230mesh, 60 Å) column (2×50 cm) equilibrated in hexane. The product waseluted sequentially with hexane and toluene, vacuum dried and weighed(34 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm) ε (M⁻¹cm⁻¹)): 780, 736, 696, 662,142000.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 735, 784.

EXAMPLE 104 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-di(2,3-naphtho)[g,q]-5,10,15,20,-tetraazoporphyrinato]siliconbis(7-oct-1-enyldimethylsilyloxide) (abbreviated as:Silicon[di(1,6-diphenylnaphthalocyanine)]dinaphthalocyaninebis(dimethylhexylvinylsilyloxide))

A mixture ofsilicon[di(1,6-diphenylnaphthalocyanine)]di-2,3-naphthalocyaninedihydroxide (25 mg) and 7-oct-1-enyldimethylchlorosilane (60 μL),imidazole (16 mg) and dimethylformamide (4 ml) was stirred at roomtemperature for 3 days. The resultant was concentrated under vacuum onthe rotary evaporator. The residue was chromatographed on a silica gel(70-230 mesh, 60 Å) column (2×50 cm) equilibrated in hexane. The productwas eluted sequentially with hexane and toluene, vacuum dried andweighed (15 mg). This compound has also been isolated as a by-productduring the chromatographic purification in Example 75.

UV-vis (tetrahydrofuran) (λ_(max)(nm) ε (M⁻¹cm⁻¹)): 786, 440000.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 792.

¹H-NMR (500 MHZ, CDCl₃): δ−2.9(S,12H); 2.0(m,4H), 1.07(m,4H),−0.06(m,4H), 0.17(m,4H), 0.6(m,4H), 1.4(m,4H), 4.7(m,4H), 5.3(m,2H),7.8(m,8H), 8.03(m,16H), 8.15(m,4H), 8.38(m,8H), 8.8 (m,4H).

EXAMPLE 105 Synthesis of[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-di(2,3-naphtho)[g,q]-5,10,15,20,-tetraazoporphyrinato]silicondihydroxide (abbreviated as:Silicon[di(1,6-diphenylnaphthalocyanine)]dinaphthalocyaninedihydroxide))

Silicon tetrachloride (600 μL) was added to a mixture of 4,9-diphenyl1,3-diiminobenz[f]isoindoline (1.0 g) and 1,3 diiminobenz[f]isoindoline(50 mg) in freshly distilled quinoline (7 ml) under an argon atmosphereand the mixture heated with stirring at 210° C. for 2 hours. Theresultant was allowed to cool, treated with water (10 ml) and refluxedfor 15 minutes. The mixture was cooled, treated with ether (20 ml) andfiltered. The organic layer of the filtrate was washed with 1 Nhydrochloric acid (2×20 ml). The solid was washed with methylenechloride (5×20 ml). The organic phases were combined and evaporated witha rotary evaporator. The residue was chromatographed on a silica gel(70-230 mesh, 60 Å) column (2×50 cm) equilibrated in methylene chloride.The product was eluted with toluene—10% isopropanol, vacuum dried andweighed (25 mg).

UV-vis (methylene chloride) (λ_(max)(nm): 794.

EXAMPLE 106 Synthesis of Silicon (Iv)phthalocyanine bis(7-oct-1-enyldimethylsilyloxide (abbreviated as: Silicon phthalocyaninebis(dimethylhexylvinyloxide))

A mixture of silicon phthalocyanine dihydroxide (500 mg),7-oct-1-enyldimethylchlorosilane (2.5 ml), imidazole (680 mg) anddimethylformamide (10 ml) was stirred at room temperature for 48 hours.The resultant was evaporated under vacuum on the rotary evaporator. Theresidue was dissolved in toluene (20 ml) and filtered. The solid washedwith toluene (40 ml). The filtrate was concentrated under vacuum on therotary evaporator and was chromatographed on a silica gel (70-230 mesh,60 Å) column (2×50 cm) equilibrated in hexane. The product was elutedsequentially with hexane and toluene, vacuum dried and weighed (324 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm) δ (M⁻¹cm⁻¹): 668, 636, 660, 283000

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 673

¹H-NMR (500 MHZ, CDCl₃,): δ −2.8(s,12H); −2.27(m,4H), −1.33(m,4H),−0.20(m,4H), 0.31(m,4H), 0.84(m,4H), 1.54(m,4H), 1.80(m,4H), 4.94(m,2H),5.75(m,2H), 8.3(m,8H), 9.65(m,8H).

EXAMPLE 107 Synthesis of Silicon (IV)phthalocyanine(10-carbomethoxydecyldimethylsilyloxide)(dimethylvinylsilyloxide)

A mixture of silicon (IV) phthalocyanine dihydroxide (500 mg), imidazole(300 mg), dimethylformamide (6 ml) and a mixture of(10-carbomethoxydecyldimethylchlorosilane (590 mg) andchlorodimethylvinylsilane (250 mg) was added and the reaction mixturestirred at room temperature for 24 hours. The resultant was concentratedunder vacuum on the rotary evaporator. The residue was chromatographedon a silica gel (70-230 mesh, 60 Å) column (2×50 cm) equilibrated inhexane. The products (a) Silicon (IV) phthalocyanine bis(10-carbomethoxydecyldimethyl silyloxide) (100 mg) and (b) silicon (IV) phthalocyanine(10-carbomethoxydecyldimethylsilyloxide)(dimethylvinylsilyloxide) (68mg) were eluted with toluene.

(a) UV-vis (tetrahydrofuran) (λ_(max)(nm): 666, 638, 602.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 671.

¹H-NMR (500 MHZ, CDCl₃): δ −2.90(s,12H), 2.27(m,4H), −1.35(m,4H), −0.22(m,4H), 0.25 (m,4H), 1.18 (m, 4H), 1.0(m,4H), 0.70 (m,4H), 1.65(m,4H),2.35(m,4H), 3.7(s,6H), 8.33(m,8H), 9.64(m,8H).

(b) UV-vis (tetrahydrofuran) (λ_(max)(nm)): 668, 636, 602.

Fluorescence (tetrahydrofuran) (λ_(max)(nm)): 673.

¹H-NMR (500 MHZ, CDCl₃): −2.9(s,6H), −2.75(s,6H), −2.27(m,4H),−1.36(m,4H), −0.015(m,4H), 0.027(m,4H), 0.07(m,4H), 0.10(m,4H),1.21(m,4H), 1.65(m,3H), 2.33(m,3H); 3.0(m,1H), 3.4(m,1H), 3.6(s),3.7(s), 4.26(m,1H); 8.33(m,8H), 9.6(m,8H).

EXAMPLE 108 Synthesis ofSulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20,-tetraazoporphyrinato]silicondihydroxide (abbreviated as: Sulfo Silicon di[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyanine dihydroxide)

A mixture of Silicondi[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyanine)dihydroxide (0.2g) and chloroform (2 ml) was stirred at room temperature for 10 minutesunder an argon atmosphere. The mixture was then cooled in an ice-bathand chlorosulfonic acid (2 ml) was added. The mixture was stirred in theice-bath for 15 minutes and then at room temperature for 20 minutes. Themixture was then refluxed for 2 hours, cooled and poured onto crushedice (100 g). The resulting green mixture was extracted with chloroform(2×30 ml). The combined organic layers were washed with water (20 ml),dried (MgSO₄) and evaporated with a rotary evaporator. The brown residuewas treated with 6 N potassium hydroxide (3 ml) with swirling and after5 minutes the mixture was partitioned between water (40 ml) and ether(20 ml). The aqueous layer was acidified with 1 N hydrochloric acid (15ml), washed with ether (40 ml) and evaporated with a rotary evaporator.The residue was vacuum dried and weighed (8 mg).

UV-vis(methanol) (λ_(max)(nm)): 650, 658, 692, 726, 748(sh).

UV-vis(water) (λ_(max)(nm)): 654, 662, 732, 758 (sh).

Fluorescence (water) (λ_(max)(nm)): 773.

IR(KBr)(cm¹): 3153, 1720, 1405, 1225, 1182, 1037, 1014, 622.

EXAMPLE 109 Synthesis of Acetylthiopropionic Acid

To a stirred solution of 3-mercaptopropionic acid (7 ml), and imidazole(5.4 g) in tetrahydrofuran (700 ml) was added, dropwise, over 15minutes, under argon, a solution of 1-acetylimidazole (9.6 g) intetrahydrofuran (100 ml). The solution was allowed to stir a further 3hours at room temperature after which time the tetrahydrofuran wasremoved under vacuum. The residue was treated with ice-cold water (18ml) and the resulting solution acidified with ice-cold concentratedhydrochloric acid (14.5 ml) to pH 1.5-2.0. The mixture was extractedwith diethyl ether (2×50 ml), the ether was washed with water (2×50 ml)and dried over MgSO₄ and evaporated. The residual crude yellow oilysolid product (10.5 g) was recrystallized from chloroform-hexane toafford 4.8 g (41% yield) acetylthiopropionic acid as a white solid witha melting point of 44°-45° C.

EXAMPLE 110 Synthesis of[2¹,2⁶,12¹,2⁶-tetraphenyldinaphtho[b,1]7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]silicon[poly(ethyleneglycol)][thiopropionyl poly(ethylene glycol)] (abbreviated as: Silicondi[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyanine[poly(ethyleneglycol)][poly(ethylene glycol)thiopropionate])

A solution of silicondi[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyanine[poly(ethyleneglycol thiopropionate] in 0.12M potassium carbonate in 80%methanol (1 ml) was allowed to stand at room temperature for 5 minutes.The pH of the solution was then adjusted to 7 by dropwise addition of asolution of 0.5 M potassium phosphate pH 7 which was made 1N inhydrochloric acid. The thiol content of the solution was estimated byEllman's method using dithionitrobenzoic acid. The title compound insolution is capable of being conjugated to ligand analogues, proteins,polypeptides and nucleic acids containing for example, maleimide oralkyliodide functional groups.

EXAMPLE 111 Synthesis of 2(2-amino-4-thiolbutanoic acidthiolactone)-bromoacetamide (abbreviated as: bromoacetyl-HCTL)

Bromoacetic acid (1.0 g), homocysteine thiolactone hydrochloride (1.1 g)and -pyridine (1.2 ml) were dissolved in anhydrous dimethylformamide (36ml) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(1.52 g) was added. The reaction was stirred at room temperature for 18hours. The solvents were removed under vacuum, ethanol (10 ml) was addedto dissolve the residue and then the ethanol was removed under vacuum.Ethanol (10 ml) was again added to dissolve the residue and was againremoved under vacuum. Water (20 ml) was added to the oil and the aqueoussolution was extracted 3 times with methylene chloride (45 ml). Thecombined organic extracts were dried over anhydrous magnesium sulfate.The solution was filtered and the solvent was removed under vacuum togive a clear oil. Diethyl ether (5 ml) was added and the resultingprecipitate was collected and washed on a fritted funnel. Theprecipitate was dried under vacuum and 1.0 g of the title compound wasrecovered.

EXAMPLE 112 Synthesis of[2-naphtho[b]-7,12,17-tribenzol[g,l,q]-5,10,15,20-tetraazoporphyrinato]silicondihydroxide (abbreviated as: Silicon(IV)[tri(phthalo)naphthalocyanine]hydroxide

Silicon tetrachloride (912 μL) was added to a mixture of1,3-diiminoisoindoline (1.0 g) and 1,3-diiminobenz[f]isoindoline (0.25g) in freshly distilled quinoline (3 ml) under an argon atmosphere andthe mixture heated with stirring at 210° C. for 2 hours. The resultantwas allowed to cool, treated with water (25 ml) and refluxed for 15minutes. The mixture was cooled, the solid filtered, washing the solidsequentially with water (3×10 ml) and ether (5×10 ml). The solid wasvacuum dried and weighed (1.5 g).

EXAMPLE 113 Synthesis of[2-naphtho[b]-7,12,17-tribenzo[g,l,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(7-oct-1-enyldimethyl silyloxide) (abbreviated as:Silicon[tri(phthalo)natphthalocyanine]bis(dimethylhexylvinylsilyloxide))

A mixture of Silicon (IV)[tri(phthalo)naphthalocyanine dihydroxide (1.0g). 7-oct-1-enyldimethylchlorosilane (3.0 ml), imidazole (0.68 g) anddimethylformamide (10 ml) was stirred at room temperature for 24 hours.The resultant was concentrated under vacuum on the rotary evaporator.The residue was chromatographed on a silica gel (70-230 mesh 60 Å)column (2×50 cm) equilibrated in hexane. The product was elutedsequentially with hexane and hexane—3% toluene, vacuum dried and weighed(11 mg).

UV-vis(methylene chloride) (λ_(max)(nm)): 716, 704, 684, 648, 618

Fluorescence (methylene chloride) (λ_(max)(nm)): 710

¹H-NMR (500 MHz, CDCl₃): δ −2.8(s,12H), −2.2(m,4H), −1.23(m,4H),−0.16(m,4H), 0.27(m,4H), 0.78(m,4H), 1.7(m,4H), 4.9(m,4H), 5.7(m,2H),7.94(m,2H), 8.3(m,6H), 8.7(m,2H), 9.6(m,6H), 10.1 (S,2H).

EXAMPLE 114 Synthesis of[2¹2⁶,12¹,12⁶-tetraphenyldinaphtho[b,1]7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]silicon[N-(2-butyrothiolactone)amidomethoxide]hydroxide(abbreviated as: Sulfo silicondi[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyanine[-2-butyrothiolactone)amidomethoxide]hydroxide

A mixture of sulfo silicondi[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyanine]dihydroxide (200mg), bromoacetyl homocysteine thiolactone (7 mg) and powdered potassiumcarbonate (180 mg), in dimethylformamide (2 ml) was stirred under argonat room temperature for 24 hours. The solvent was evaporated with arotary evaporator, the residue treated with ethanol (2 ml) and filteredwashing with ethanol (2 ml). The filtrate was evaporated, and theproduct vacuum dried and weighed (200 mg). This product was used withoutfurther purification in the next step.

EXAMPLE 115 Synthesis ofSulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]silicon[N-(cysteine)amidomethoxide]hydroxide(abbreviated as: Sulfo silicondi[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyanine[N-(cysteine)amidomethoxide]hydroxide)

A solution of sulfo silicondi[(1,6-diphenyl-2,3-naphthalocyanine]diphthalocyanine[N-(2-butyrothiolactone)amidomethoxide]hydroxide(10 mg) in water (182 ml) was treated with 1 N potassium hydroxidesolution (46 ml) and allowed to stand at room temperature for 10minutes. The pH of the solution was then adjusted to 7 by dropwiseaddition of a solution of 0.5 M potassium phosphate pH 7 which was made1 N in hydrochloric acid. The thiol content of the solution wasestimated by Ellman's method using dithionitrobenzoic acid. The titlecompound in solution is capable of being conjugated to ligand analogues,proteins, polypeptides and nucleic acids containing, for example,maleimide or alkyliodide functional groups.

EXAMPLE 116 Synthesis of Silicon tetra-tert-butylphthalocyaninebis[(4-aminobutyl)dimethylsilyloxidel

To a stirred solution of silicon tetra-tert-butyl phthalocyaninedihydroxide (800 mg) in pyridine (140 ml) was added4-aminobutyldimethylmethoxysilane (950 μL). The solution was heated toreflux and pyridine allowed to distill off until 50 ml of distillate hadbeen collected. The solution was allowed to cool and the residualpyridine removed under vacuum. The residue was chromatographed on asilica gel (70-230 mesh, 60 Å, 3×50 cm) column equilibrated in methylenechloride. The product was eluted sequentially with methylene chloride,tetrahydrofuran and finally tetrahydrofuran—2% triethylamine. The darkblue product was vacuum dried and weighed (355 mg).

UV-vis (tetrahydrofuran) (λ_(max)(nm)): 606, 644, 672.

EXAMPLE 117 Synthesis ofSulfo[2¹,2⁶12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20,-tetraazoporhyrinato]silicondihydroxide (abbreviated as: Sulfo silicondi[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyanine dihydroxide)

Silicon di[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyaninedihydroxide (110 mg) was dissolved in (1 ml) concentrated sulfuric acid,and 10 minutes later chlorosulfonic acid (150 ml) was added. Thereaction mixture was then heated in an oil bath (100-130° C.) for 2.5hours. The reaction mixture was allowed to cool to room temperature andpoured onto crushed ice (30 g). The pH of the green solution wasadjusted with solid potassium carbonate to pH=9.0. The solvent wasevaporated with a rotary evaporator. The residue was dissolved in 200 mMpotassium phosphate buffer (pH=7.0) and applied to a C₁₈-column (12cm×2.5 cm) that was equilibrated in 200 mM potassium phosphate buffer(pH=7.0). The column was washed with 200 mM potassium phosphate buffer(pH=7.0) (50 ml) water (300 ml), and the product was eluted with amixture of water and methanol 2:1 (v/v). The solvent was evaporated witha rotary evaporator. The residue was vacuum dried and weighed (137 mg).

UV-vis (Water) (λ_(max)(nm)) 658, 698, 732, 756(sh).

UV-vis (Methanol) (λ_(max)(nm)) in MeOH=648, 688, 724, 742(sh).

IR(KBr)(cm⁻¹) 3629, 3465, 3065, 2593, 1721, 1622, 1521, 1422, 1353,1335, 1284, 1194, 1088, 1039, 1013, 941, 906, 821, 760, 651, 620.

¹H-NMR (500 MHz, DMSO-d₆) δ=−2.4(s, OH), 8.1(m, Ar —H).

EXAMPLE 118 Synthesis ofSulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(4-Aminobutyldimethysilane) (abbreviated as: Sulfo silicondi[(1,6-diphenyl)-2,3-naphthalocyanine)phthalocyanine]bis(4-Aminobutyldimethylsilane)

To a suspension of sulfo silicondi[(1,6-diphenyl-2-3-naphthalocyanine)phthalocyanine]dihydroxide (32 mg)in pyridine (20 ml) was added 4-aminobutyldimethylmethoxysilane (50 ml),and the reaction mixture was heated in an oil bath (140° C.) for 3hours. The reaction mixture was allowed to cool to room temperature, andDMF (5 ml) was added followed by 4-aminobutyl-dimethylethoxysilane (100ml). The reaction mixture was then refluxed for 16 hours. After coolingthe solvent was evaporated with a rotary evaporator. The residue wasdissolved in methanol (2 ml) and applied on a C18 column. The column waswashed with (200 mM) potassium phosphate buffer pH=7.0 (20 ml), water(200 ml), water/methanol=3:1 (v/v) (40 ml), water/methanol=2:1 (v/v) (40ml). The product was eluted with 95% methanol, the solvent wasevaporated with a rotary evaporator, and the product was dried undervacuum and weighed (32 mg).

UV-vis (Water) (λ_(max)(nm)) 658, 696(sh), 730.

UV-vis (Methanol) (λ_(max)(nm)) 648, 686, 722, 748(sh).

EXAMPLE 119 Synthesis ofSulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(3-amino-propyldiisopropylsilyloxide)(abbreviated as: Sulfo silicondi[(1,6-diphenyl)-2,3-naphthalocyanine)phthalocyanine]bis-(3-amino-propyldiisopropylsilyloxide)

A mixture of sulfo silicondi[(1,6-diphenyl)-2-3-naphthalocyanine]phthalocyanine dihydroxide (50mg) 3-amino-propyldiisopropylmethoxysilane (190 microliters) in toluene(2 ml) was refluxed for 16 hours. After cooling to room temperature thesolvent was evaporated with a rotary evaporator. The green oily residuewas applied to a C₁₈ column. The column was washed with (200 mM)phosphate buffer (pH=7.0) (50 ml), water (200 ml), water/methanol[(3:1;(v/v) (20 ml)], water/methanol 2:1 (v/v). The product was elutedwith 95% methanol. The solvent was evaporated with a rotary evaporator,and the residue was vacuum dried and weighed (40.0 mg).

UV-vis (Methanol) (λ_(max)(nm)): 648, 686, 724, 744(sh).

EXAMPLE 120 Synthesis ofSulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(10-carbomethoxydecyl)dimethylsilyloxidel (abbreviated as: SulfoSilicon di[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyaninebis-[(10-carbomethoxydecyl)dimethylsilyloxidel

A mixture of imidazole (33 mg) and(10-carbomethoxydecyl)dimethylchlorosilane in (1.0 ml) pyridine wasstirred for 1 hour at room temperature, and sulfo silicondi[(1,6-diphenyl)-2-3-naphthalocyanine]diphthalocyanine dihydroxide (20mg) in pyridine (3 ml) was added. After stirring the reaction mixturefor 16 hours, the pyridine was evaporated with a rotary evaporator. Theresidue was triturated with (2 ml) (200 mM) potassium phosphate buffer,PH=7.0 (2 ml) (200 mM) and applied to C₁₈ column (equilibrated with (200mM) potassium phosphate buffer pH=7.0). The column was washed withpotassium phosphate buffer (60 ml) (200 mM) (pH 7.0), water (210 ml),water/MeOH [(1:1;(v/v) (40 ml)], and water/MeOH [(1:2; (v/v) (35 ml)].The product was then eluted with 95% methanol, the solvent wasevaporated with a rotary evaporator. The residue was vacuum dried andweighed (8 mg).

UV-vis (Water) (λ_(max)(nm)) 658, 694, 730, 750,(sh).

UV-vis (Methanol) (λ_(max)(nm)) 650, 690, 726, 746(sh).

IR(KBr)(cm⁻¹) 2924, 2854, 1744.

Fluorescence (methanol) λ_(max)(nm): 752

Fluorescence (water) λ_(max)(nm): 761

EXAMPLE 121 Synthesis ofSulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(7-oct-1-enyldimethylsilyloxide) (abbreviated as: Sulfo Silicondi[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyaninebis-[(7-oct-1-enyldimethylsilyloxide)

A mixture of sulfo silicondi[(1,6-diphenyl)-2-3-naphthalocyanine]diphthalocyanine dihydroxide (10ml) and imidazole (41 mg) in dimethylformamide (2 ml) was stirred atroom temperature for 10 minutes and 7-oct-1-enyldimethylchlorosilane wasadded. The mixture was stirred for 14 hours at room temperature and thesolvent was moved with a rotary evaporator. The residue was trituratedwith (2 ml) (200 mM potassium phosphate buffer pH=7.0 and applied to aC₁ column (equilibrated with 200 mM potassium phosphate buffer, pH=7.0).The column was washed with potassium phosphate buffer (40 ml), water(150 ml) and water/methanol (2:1 (v/v)). The product was eluted with 95%methanol, and the solvent was evaporated with a rotary evaporator. Theresidue was vacuum dried and weighed (9 mg).

¹H-NMR (500 MHz, DMSO) δ −2.8(s,12H), −2.1(m,4H), −1.3(m,4H),−0.23(m,4H) 0.06(m,4H), 0.5(m,4H), 1.3(m,4H), 4.7(m,4H), 5.4(m,2H),8.0(Ar—H).

EXAMPLE 122 Synthesis of Sulfo silicon naphthalocyaninebis(4-aminobutyldimethyl silyloxide

A mixture of sulfo silicon naphthalocyanine dihydroxide triethylammonium salt (30 mg) and pyridine was stirred at room temperature for10 minutes, and then N,N-Diisopropylethylamine (10 ml) followed by4-aminobutyldimethylmethoxysilane (380 microliters) were added. Thereaction mixture was heated in an oil bath for 2 hours at 130° C. Aftercooling to room temperature the solvent was removed with a rotaryevaporator and the residue was triturated with 200 mM potassiumphosphate buffer pH=7.0 (2 ml) and applied to a C₁₈ column (1.5×23 cmfilled with C18 to 7.0 cm height). The column was washed with 200 mMpotassium phosphate buffer (40 ml), water (80 ml), water/methanol (2:1)(40 ml), water/methanol (2:1) (70 ml), and the major green fraction waseluted with water/methanol (1:3) (40 ml). The solvent was removed with arotary evaporator and the residue was vacuum dried and weighed (14 mg).

IR(KBr)(cm⁻¹) 3069, 2964, 1631, 1528, 1362, 1252, 1184, 1091, 1067,1035, 844, 798, 761, 728, 691, 615.

¹H-NMR (500 MHz, DMSO) δ −2.5(S,12H), −1.9(m,4H), −1.0(m,4H), 0.4(m,4H),2.0(m,4H).

EXAMPLE 123 Synthesis of Sulfo silicon naphthalocyaninebis[10-(carbomethoxy)decyldimethylsilyloxide]

To a stirred solution of imidazole (109 mg) in pyridine (2 ml) was added10-(carbomethoxy)decyldimethylchlorosilane (513 microliters), and themixture stirred for 20 min. at room temperature. Sulfo siliconnaphthalocyanine dihydroxide (60 mg)(neat) was then added followed bypyridine (1 ml) and 10-(carbomethoxy)decyldimethylchlorosilane (0.6 ml).The reaction mixture was allowed to stir 14 hours, and the solventevaporated with a rotary evaporator. The residue was suspended in 40 mMpotassium phosphate buffer (pH 7.0) (2 ml) and chromatographed on a C¹⁸column. After washing the column with 200 mM potassium phosphate buffer(40 ml) and water (300 ml), the product was eluted with water/methanol(1:1). The solvent was evaporated with a rotary evaporator. The residuewas vacuum dried and weighed (55 mg).

EXAMPLE 124 Synthesis of sulfo silicon naphthalocyaninebis(3-aminopropyldiisopropylsilane)

A mixture of sulfo silicon naphthalocyanine (50 mg) and3-aminopropyldiisopropylethoxysilane (200 ml) in toluene (3 ml) isrefluxed for 16 hours. The reaction mixture is allowed to cool to roomtemperature and the solvent is evaporated with a rotary evaporator. Theresidue can be purified on a C₁₈ column, with (200 mM) potassiumphosphate buffer, (pH=7.0) water and 95% methanol.

EXAMPLE 125 Synthesis of 1,4-diphenylnaphthalene-2,3-di-carbonitrile

In a dry 2 L 3-necked round bottom flask equipped with a magneticstirring bar, dropping funnel, gas inlet tube attached to an argon gascylinder, was placedtetrahydro-1,4-diphenyl-1,4-epoxy-naphthalene-2,3-dicarbonitrile (20 g)and dry tetrahydrofuran (450 ml) while purging the flask with argon gas.The mixture was stirred for 20 minutes. The flask was cooled to −78° C.(acetone/dry ice) and Lithium bis(trimethylsilyl)-amide (150 ml, 1.0 MTHF) was added dropwise over 2 hours. The mixture was allowed to stir atthis temperature, and saturated ammonium chloride (300 ml) was added.The mixture was allowed to warm to room temperature, and the white solidwas filtered off. The organic layer of the filtrate was separated. Theaqueous layer was washed with ether (100 ml). The combined organiclayers were dried (magnesium sulfate). After the magnesium sulfate wasfiltered off, the solvent was evaporated with a rotary evaporator, theresidue triturated with ether, and the solid filtered, dried undervacuum and weighed (17 g).

IR(KBr)(cm⁻¹) 3059, 2232, 1608, 1494, 1446, 1400, 1378, 1183, 1077,1029, 1001, 931, 796, 783, 757, 706, 681, 657, 620, 517, 437.

¹H-NMR (500 MHz, DMSO) δ 7.5(m,4H), 7.6(m,8H), 7.8(m,2H).

EXAMPLE 126 Synthesis ofSulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis[N-succinamido)aminobutyldmethyl silyloxide

A mixture of sulfo silicondi[(1,6-diphenyl)-2,)-naphthhalocyanine]diphthalocyaninebis(4-aminobutyldimethylsilyloxide) (20 mg) and succinic anhydride (50mg) in dimethylformamide (4 ml) is refluxed for 2 hours. The reactionmixture is allowed to cool to room temperature and the solvent isevaporated with a rotary evaporator. The residue can be purified on aC₁₈ column, with (200 mM) potassium phosphate buffer, (pH 7.0). waterand methanol.

EXAMPLE 127 Synthesis ofSulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis[4[(acetylthiopropionamido)butyl]dimethylsilyloxidel (Abbreviated as:Sulfo Silicon di[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyaninebis((acetylthiopropionamido)butyl silyloxide)

A mixture of sulfo silicondi[(1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyaninebis(4-aminobutyldimethylsilyloxide) in dimethylformamide and a solutionof acetylthioproponic acid and 1,1′-Carbonyldiimidazole indimethylformamide is stirred at room temperature for 1 hour. The solventis evaporated with a rotary evaporator. The residue can be purified on aC₁₈ column, with (200 mM) potassium phosphate buffer (pH 7.0) water andmethanol.

EXAMPLE 128 Synthesis ofSulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis[4[(thiopropionamido)butyl]dimethylsilyloxide](Abbreviated as: SulfoSilicon di[1,6-diphenyl)-2,3-naphthalocyanine]diphthalocyaninebis((thiopropionamido)butyl dimethyl silyloxide)

A mixture of sulfo silicondi[(1,6-diphenyl-2,3-naphthalocyanine]diphthalocyaninebis((acetylthiopropionamido)butyl dimethyl silyloxide) in 50% (v/v)aqueous methanol (20 mM) and potassium carbonate at 200 mM is stirred atroom temperature for 20 min. The mixture is neutralized to pH 7 with 1 Nhydrochloric acid and the solvent is evaporated with a rotaryevaporator. The residue can be purified on a C18 column, with (200 mM)potassium phosphate buffer (pH 7.0), water and methanol.

EXAMPLE 129 Preparation of a Conjugate of Sulfonated HybridPhthalocyanine Derivative and an Antibody

A monoclonal antibody against human chorionic gonadotropin (Calbiochem,San Diego, Calif.) at 10 mg/ml in 50 mM potassium phosphate, 150 mMsodium chloride, pH 7.0, is reacted with SMCC (Pierce Chemical Co.,Rockford, Ill.) at 0.6 mM at room temperature for 1.5 h. Theantibody-maleimide is purified on a column of Sephadex G-25 equilibratedin 50 mM potassium phosphate, 150 mM sodium chloride, pH 7.0. Thepurified antibody-maleimide (2.5 ml) at 5 mg/ml is reacted with anexcess of sulfo silicondi[(1,6-diphenyl-2,3-naphthalocyanine]diphthalocyaninebis((thiopropionamido)butyl dimethylsilyloxide) (2.5 ml) at 0.6 mM atroom temperature for 3 h. A solution of N-ethyl maleimide in water isthen added to a final concentration of 3 mM and the solution is stirredfor an additional 30 min. The antibody-hybrid phthalocyanine derivativeis purified on a Sephadex G-25 column equilibrated in 50 mM potassiumphosphate, 150 mM sodium chloride, 10 mg/ml bovine serum albumin, pH7.0.

EXAMPLE 130 Preparation of a Conjugate of Sulfonated HybridPhthalocyanine Derivative and a Ligand Analogue

In one embodiment the ligand analogue is morphine. Morphine-HCTL (seeU.S. Pat. No. 5,089,391, example 4, incorporated by reference) ishydrolyzed in 0.12 M potassium carbonate/40% (v/v) aqueous methanol at20 mM at room temperature for 5 min. The solution is then adjusted to pH7.0 with 1 N hydrochloric acid and diluted to 5 mM with 50 mM potassiumphosphate, pH 7.0. A homobifunctional cross linker,(bis-maleimidohexane, Pierce Chemical Co., Rockford, Ill.) in 50 mMpotassium phosphate, pH 7.0, is added to a final concentration of 50 mM.The solution is stirred at room temperature for 1 h and themorphine-maleimide derivative is purified on a reversed phase C₁₈ columnusing a linear gradient of 50 mM potassium phosphate, pH 7 and methanol.The morphine-maleimide solution in 50 mM potassium phosphate, pH 7.0, isadded to a solution of sulfo silicondi[(1,6-diphenyl-2,3-naphthalocyanine]diphthalocyaninebis((thiopropionamido)butyl dimethylsilyloxide) in 50 mM potassiumphosphate, pH 7.0, so that the final concentrations are 10 mM and 2 mM,respectively. The solution is stirred at room temperature for 3 h andthe sulfonated hybrid phthalocyanine-morphine derivative is purified ona reversed phase C₁₈ column using a linear gradient of 10 mM potassiumphosphate, pH 7.0 and methanol.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aformulation” includes mixtures of different formulations and referenceto “the method of treatment” includes reference to equivalent steps andmethods known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar to equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to describe and disclose specificinformation for which the reference was cited in connection with.

1. A water soluble hybrid phthalocyanine derivative.
 2. A derivative ofclaim 1 wherein the derivative is silicon[di(1,6-diphenyl-2,3naphthalocyanine)]diphthalocyanine bis(poly(ethylene glycol)methylether].
 3. A derivative of claim 1 wherein the derivative issilicon[di(1,6-diphenyl-2,3-naphthalocyanine)]diphthalocyaninebis[poly(ethylene glycol)].
 4. A derivative of claim 1 wherein thederivative issilicon[di(1,6-diphenyl-2,3-naphthalocyanine)]diphthalocyanine[poly(ethyleneglycol)][poly(ethylene glycol)acetylthiopropionate].
 5. A derivative ofclaim 1 wherein the derivative is silicon[di(1,6-diphenyl2,3-naphthalocyanine)]di(2,3-dicarboxyphthalocyanine)dihydroxide.
 6. Aderivative of claim 1 wherein the derivative is silicon[di(1,6-diphenyl2,3-naphthalocyanine)]di(2,3-dicarboxyphthalocyanine)bis[poly(ethyleneglycol)methyl ether].
 7. A derivative of claim 1 wherein the derivativeis sulfo silicon di[(1,6-diphenyl-2,3-naphthalocyanine]diphthalocyaninedihydroxide.
 8. A derivative of claim 1 wherein the derivative issilicon[di(1,6-diphenyl-2,3-naphthalocyanine)]diphthalocyanine[poly(ethyleneglycol)][poly(ethylene glycol)thiopropionate].
 9. A derivative of claim1 wherein the derivative is sulfo silicondi[(1,6-diphenyl-2,3-naphthalocyanine]diphthalocyanine[-2-butyrothiolactone)amidomethoxide]hydroxide.10. A derivative of claim 1 wherein the derivative is sulfo silicondi[(1,6-diphenyl-2,3-naphthalocyanine]diphthalocyanine[N-(cysteine)amidomethoxide]hydroxide.11. A derivative of claim 1 wherein the derivative is silicontetra-tertbutylphthalocyanine bis[(4-aminobutyl)dimethylsilyloxide]. 12.A derivative of claim 1 wherein the derivative issulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]silicondihydroxide.
 13. A derivative of claim 1 wherein the derivative issulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(4-Aminobutyldimethylsilyloxide)
 14. A derivative of claim 1 whereinthe derivative issulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(3-amino-propyldiisopropylsilyloxide).
 15. A derivative of claim 1wherein the derivative issulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis-[(10-carbomethoxydecyl)dimethyl silyloxide].
 16. A derivative ofclaim 1 wherein the derivative issulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis(7-oct-1-enyldimethylsilyloxide).
 17. A derivative of claim 1 whereinthe derivative is sulfo silicon naphthalocyaninebis(4-aminobutyldimethyl silyloxide).
 18. A derivative of claim 1wherein the derivative is sulfo silicon naphthalocyaninebis[10-(carbomethoxy)decyl dimethylsilyloxide].
 19. A derivative ofclaim 1 wherein the derivative is sulfo silicon naphthalocyaninebis(3-aminopropyldiisopropylsilyloxide).
 20. A derivative of claim 1wherein the derivative issulfo[2¹,2⁶,12¹,12⁶-tetraphenyldinaphtho[b,l]-7,17-dibenzo[g,q]-5,10,15,20-tetraazoporphyrinato]siliconbis[N-succinamido)aminobutyldimethyl silyloxide.
 21. A method fordetermining the presence or amount of an analyte of interest, saidmethod comprising: forming a reaction mixture comprising said analyteand a population of fluorescent particles comprising receptor specificfor said analyte; capturing fluorescent particles bound to said analyteon a solid phase; and generating a fluorescent signal from capturedfluorescent particles, wherein said fluorescent particles comprise: afirst compound selected from the group consisting of siliconphthalocyanine bis(dimethylhexylvinylsilyloxide) and siliconphthalocyanine bis(trihexylsilyloxide); and a second compound that is abis(dimethylhexylvinylsilyloxide)-substituted orbis(trihexylsilyloxide)-substituted phthalocyanine, naphthalocyanine, oranthranylocyanine derivative, or abis(dimethylhexylvinylsilyloxide)-substituted orbis(trihexylsilyloxide)-substituted hybrid phthalocyanine derivative,wherein the size of said fluorescent particle is between 0.1 nm and 5000nm, and wherein said first compound differs in structure from saidsecond compound.
 22. The method according to claim 21, wherein saidfluorescent particle comprises a latex matrix.
 23. The method accordingto claim 21, wherein said fluorescent particle comprises a silicamatrix.
 24. The method according to claim 21, wherein said firstcompound and said second compound are eachbis(dimethylhexylvinylsilyloxide)-substituted compounds.
 25. The methodaccording to claim 21, wherein said first compound is siliconphthalocyanine bis(dimethylhexylvinylsilyloxide), and said secondcompound is silicon 2,3-naphthalocyaninebis(dimethylhexylvinylsilyloxide).
 26. The method according to claim 21,wherein said first compound is silicon phthalocyaninebis(dimethylhexylvinylsilyloxide), and said second compound is siliconphthalocyanine bis(trihexylsilyloxide).
 27. The method according toclaim 21, wherein said first compound is silicon phthalocyaninebis(dimethylhexylvinylsilyloxide), and said second compound is silicon[di(1,6-diphenyl-2,3-naphthalocyanine)](2,3-naphthalocyanine)phthalocyaninebis(dimethylhexylvinylsilyloxide).
 28. The method according to claim 21,wherein said first compound is silicon phthalocyaninebis(dimethylhexylvinylsilyloxide), and said second compound is silicon[di(1,6-diphenyl-2,3-naphthalocyanine)][di(2,3-tert-butylphthalocyanine]bis(dimethylhexylvinylsilyloxide).29. The method according to claim 21, wherein said first compound issilicon phthalocyanine bis(dimethylhexylvinylsilyloxide), and saidsecond compound is silicon[di(2,3-naphthalocyanine)][di(1,4-diphenylphthalocyanine]bis(dimethylhexylvinylsilyloxide).30. The method according to claim 21, wherein said first compound issilicon phthalocyanine bis(dimethylhexylvinylsilyloxide), and saidsecond compound is silicon[di(1,6-diphenyl-2,3-naphthalocyanine)]diphthalocyaninebis(dimethylhexylvinylsilyloxide).
 31. The method according to claim 21,wherein said first compound is silicon phthalocyaninebis(dimethylhexylvinylsilyloxide), and said second compound is silicon[di(1,6-diphenyl-2,3-naphthalocyanine)][di(2,3-dicyanophthalocyanine)]bis(dimethylhexylvinylsilyloxide).32. The method according to claim 21, wherein said first compound issilicon phthalocyanine bis(dimethylhexylvinylsilyloxide), and saidsecond compound is silicon 2,3-naphthalocyaninebis(dimethylhexylvinylsilyloxide).
 33. The method according to claim 21,wherein said first compound is silicon phthalocyaninebis(dimethylhexylvinylsilyloxide), and said second compound is silicon[di(1,6-diphenylnaphthalocyanine)]diphthalocyaninebis(dimethylhexylvinylsilyloxide).
 34. The method according to claim 21,wherein said receptor specific for said analyte is an antibody.
 35. Themethod according to claim 21, wherein said receptor specific for saidanalyte is a nucleic acid.
 36. The method according to claim 21, whereinthe size of said fluorescent particle is between 1 nm and 1000 nm. 37.The method according to claim 21, wherein said signal is generated byexciting said fluorescent particles at a wavelength above 600 nm anddetecting a fluorescent signal generated thereby.
 38. The methodaccording to claim 21, wherein said analyte in said reaction mixture isfrom a biological fluid.
 39. The method according to claim 38, whereinsaid biological fluid is urine, blood, serum, or plasma.